Nanoparticle delivery vehicle

ABSTRACT

A nanoparticle delivery vehicle, comprising a nanoparticle, an active agent and a nuclear localization signal and methods of modulating gene expression and protein expression employing the nanoparticle delivery vehicle. A representative method includes providing a nanoparticle delivery vehicle comprising a nanoparticle having a diameter of about 30 nm or less, an active agent and a nuclear localization signal; and contacting a target cell with the nanoparticle delivery vehicle, whereby an active agent is delivered to the nucleus of a target cell. Another representative method includes providing a nanoparticle delivery vehicle comprising a nanoparticle having a diameter greater than or equal to about 30 nm, an active agent and a nuclear localization signal; and contacting a target cell with the nanoparticle delivery vehicle, whereby an active agent is delivered to the cytoplasm of a cell.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on and claims priority to U.S. ProvisionalPatent Application Ser. No. 60/304,236, filed Jul. 10, 2001, hereinincorporated by reference in its entirety.

GRANT STATEMENT

This work was supported by NSF grants NSF-DMR (9900073) and NSF-MDB(9874895). Thus, the U.S. Government has certain rights in theinvention.

TECHNICAL FIELD

The present invention relates to compositions for and methods ofdelivering an active agent to and into cells. More particularly, themethod employs a nanoparticle delivery vehicle as a vehicle for carryingproteins, nucleic acids, protein and nucleic acid analogs, smallmolecules and other compounds to the surface of a cell, into thecytoplasm of a cell or into a cell's nucleus.

Table of Abbreviations ATP adenosine triphosphate ADP adenosinediphosphate AS antisense AS-ODN antisense oligodeoxynucleotides bipybipyridine cDNA complementary DNA DNA deoxyribonucleic acid dsDNA doublestranded DNA EDTA ethylenediaminetetraacetic acid HEPESN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid ITO indium tin oxideIV intravenous kDa kilodalton(s) LB Luria broth MES2-[N-Morpholino]ethanesulfonic acid mRNA messenger RNA NDP nucleotidediphosphate NLS nuclear localization signal nt nucleotide NTP nucleotidetriphosphate ODN oligodeoxynucleotide PACVD plasma-assisted chemicalvapor deposition PAGE polyacrylamide gel electrophoresis PBS phosphatebuffered saline PCR polymerase chain reaction pl isoelectric point PNApeptide nucleic acid analog RES reticuloendothelial system RME receptormediated endocytosis RNA ribonucleic acid SDS sodium dodecyl sulfateSDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis ssDNAsingle stranded DNA TEM transmission electron microscopy Amino AcidAbbreviations Single-Letter Code Three-Letter Code Name A Ala Alanine VVal Valine L Leu Leucine I Ile Isoleucine P Pro Proline F PhePhenylalanine W Trp Tryptophan M Met Methionine G Gly Glycine S SerSerine T Thr Threonine C Cys Cysteine Y Tyr Tyrosine N Asn Asparagine QGln Glutamine D Asp Aspartic Acid E Glu Glutamic Acid K Lys Lysine R ArgArginine H His Histidine

BACKGROUND ART

The development of new forms of therapeutics that use macromoleculessuch as proteins or nucleic acids as therapeutic agents has created aneed to develop new and effective approaches of delivering suchmacromolecules to their appropriate cellular targets. Therapeutics basedon either the use of specific polypeptide growth factors or specificgenes to replace or supplement absent or defective genes are examples oftherapeutics that might require such new delivery systems. Therapeuticsinvolving oligonucleotides that interact with DNA to modulate theexpression of a gene or other segment of DNA might also require a newdelivery system. Clinical application of such therapies depends not onlyon the reliability and efficiency of new delivery systems but also ontheir safety and on the ease with which the technologies underlyingthese systems can be adapted for large-scale pharmaceutical production,storage, and distribution of the therapeutic formulations.

Gene therapy has become an increasingly important mode of treatingvarious genetic disorders. The potential for providing effectivetreatments, has stimulated an intense effort to apply this technology todiseases for which there have been no effective treatments. Recentprogress in this area has indicated that gene therapy can have asignificant impact not only on the treatment of single gene disorders,but also on other more complex diseases such as cancer. However, asignificant obstacle in the attainment of efficient gene therapy regimehas been the difficulty of designing new and effective approaches fordelivering therapeutic nucleic acids to cells and intracellular targets.Indeed, an ideal vehicle for the delivery of nucleic acids or proteinsinto cells and tissues should be highly efficient, safe to use, easy toproduce in large quantity and have sufficient stability to bepracticable as a pharmaceutical delivery vehicle.

When nucleic acids are used as “active agents” in a gene therapy regime,there are essentially two systems based on viral vectors or nonviralvectors that are described in the art: (1) retro, adeno and herpesviruses (or their recombinants) are presently being studied in vivo asviral vectors; and (2) liposomes and ligands of cell surface-specificreceptors are being researched in vivo as nonviral vectors (Wu & Wu,(1991) Biotherapy 3: 87-95; Ledley, (1993) Clin. Invest. Med. 16:78-88). Nanocrystalline particles are also being investigated (U.S. Pat.No. 5,460,831 to Kossovsky). All of these approaches suffer from avariety of disadvantages, including undesired in vivo degradation and alack of specificity for a given target structure, for example, thenucleus of a cell or the surface of a cell expressing a particular typeof structure.

Nanoparticle technology has found application in a variety ofdisciplines, but has found minimal application in pharmacology and drugdelivery. The development of therapeutic nanoparticles was firstattempted around 1970, and the proposed nanoparticles were intended tofunction as carriers of anticancer and other drugs (Couvreur et al.,(1982) J. Pharm. Sci., 71: 790-92). Attempts were also made to elucidatemethods by which the uptake of the nanoparticles by the cells of thereticuloendothelial system (RES) would be minimized (Couvreur et al.,(1986) in Polymeric Nanoparticles and Microspheres, (Guiot & Couvreur,eds.), CRC Press, Boca Raton, pp. 27-93). Other attempts pursued the useof nanoparticles for treatment of specific disorders. See, e.g.,Labhasetwar et al., (1997) Adv. Drug. Del. Rev., 24: 63-85.

Although nanoparticles have shown promise as useful tools for drugdelivery systems, many problems remain. Some unsolved problems relate tothe control, selection, and behavior of various particle sizes, as wellas problems surrounding the loading of particles with therapeutics.Additionally, the targeting of the nanoparticle to the appropriatecellular site has remained problematic. The design and provision of ananoparticle delivery vehicle that addresses these problems thusrepresents and ongoing and long-felt need in the art.

SUMMARY OF THE INVENTION

A nanoparticle delivery vehicle is disclosed. In one embodiment, thenanoparticle delivery vehicle comprises: (a) a nanoparticle; (b) anactive agent; and (c) a nuclear localization signal. In anotherembodiment, the nanoparticle delivery vehicle comprises (a) a pluralityof targeting agents; (b) a nanoparticle scaffold; and (c) an activeagent.

Preferably, the active agent is selected from the group consisting ofdouble stranded nucleic acids, single stranded nucleic acids, chemicallymodified nucleic acids, peptide nucleic acids, proteins and smallmolecules. Preferably, the nanoparticle delivery vehicle furthercomprises a tether sequence attached to, and disposed between, theactive agent and the nanoparticle. Preferably, the nanoparticle deliveryvehicle further comprises a cell surface recognition sequence.Preferably, the nanoparticle delivery vehicle is disposed in apharmaceutically acceptable diluent. Preferably, the nanoparticledelivery vehicle further comprises a detectable moiety.

Optionally, the nanoparticle delivery vehicle can further comprise twoor more different active agents. Also optionally, the nanoparticledelivery vehicle can further comprise a biocompatibility-enhancingagent. As a further option, the nanoparticle delivery vehicle canfurther comprise a protective coating covering at least part of thedelivery vehicle. In one embodiment, the protective coating can coverthe entire delivery vehicle, including the active agent(s) and targetingagent(s). The protective coating can comprise a polymer. The protectivecoating can also comprise a biological material. The biological materialcan be a protein, lipid, carbohydrate, or combination thereof.

A method of delivering an active agent to the nucleus of a cell isdisclosed. The method comprises:(a) providing a nanoparticle deliveryvehicle of the present invention comprising a nanoparticle having adiameter of about 30 nm or less; and (b) contacting a target cell withthe nanoparticle delivery vehicle, whereby an active agent is deliveredto the nucleus of a target cell. Preferably, the active agent isselected from the group consisting of double stranded nucleic acids,single stranded nucleic acids, chemically modified nucleic acids,peptide nucleic acids, proteins and small molecules. Preferably, thenanoparticle delivery vehicle further comprises a tether sequenceattached to, and disposed between, the active agent and thenanoparticle. Preferably, the nanoparticle delivery vehicle furthercomprises a cell surface recognition sequence. Preferably, thenanoparticle delivery vehicle is disposed in a pharmaceuticallyacceptable diluent. Preferably, the nanoparticle delivery vehiclefurther comprises a detectable moiety.

A method of delivering an active agent to the cytoplasm of a cell isdisposed. The method comprises: (a) providing a nanoparticle deliveryvehicle of the present invention comprising a nanoparticle having adiameter greater than or equal to about 30 nm; and (b) contacting atarget cell with the nanoparticle delivery vehicle. Preferably, theactive agent is selected from the group consisting of double strandednucleic acids, single stranded nucleic acids, chemically modifiednucleic acids, peptide nucleic acids, proteins and small molecules.Preferably, the nanoparticle delivery vehicle further comprises a tethersequence attached to, and disposed between, the active agent and thenanoparticle. Preferably, the nanoparticle delivery vehicle furthercomprises a cell surface recognition sequence. Preferably, thenanoparticle delivery vehicle is disposed in a pharmaceuticallyacceptable diluent. Preferably, the nanoparticle delivery vehiclefurther comprises a detectable moiety.

A method of modulating the expression of a target nucleic acid sequenceis disclosed. The method comprises:(a) providing a nanoparticle deliveryvehicle of the present invention comprising an active agent capable ofinteracting with a target nucleic acid sequence whose expression is tobe modulated; (b) contacting a target cell comprising a target nucleicacid sequence with the nanoparticle delivery vehicle; and (c) modulatingthe expression of the target nucleic acid sequence through thecontacting of step (b). Preferably, the nanoparticle delivery vehiclefurther comprises a tether sequence attached to, and disposed between,the active agent and the nanoparticle. Preferably, the nanoparticledelivery vehicle further comprises a cell surface recognition sequence.Preferably, the nanoparticle delivery vehicle is disposed in apharmaceutically acceptable diluent. Preferably, the nanoparticledelivery vehicle further comprises a detectable moiety.

A method of modulating the expression of a target protein is disclosed.The method comprises: (a) providing a nanoparticle delivery vehicle ofthe present invention comprising a single stranded antisense nucleicacid sequence complementary to a nucleic acid sequence encoding a targetprotein; (b) contacting a target cell comprising a nucleic acid sequenceencoding a target protein with the nanoparticle delivery vehicle; and(c) modulating the expression of the target protein through thecontacting of step (b). Preferably, the nanoparticle delivery vehiclefurther comprises a tether sequence attached to, and disposed between,the active agent and the nanoparticle. Preferably, the nanoparticledelivery vehicle further comprises a cell surface recognition sequence.Preferably, the nanoparticle delivery vehicle is disposed in apharmaceutically acceptable diluent. Preferably, the nanoparticledelivery vehicle further comprises a detectable moiety.

A method of modulating transcription in a sample is disclosed. Themethod comprises: (a) providing a nanoparticle delivery vehicle of thepresent invention comprising an active agent comprising a ligand forwhich a wild-type transcription component has greater affinity than anatural ligand of the wild-type transcription component; (b) contactinga sample comprising the wild-type transcription component with thenanoparticle delivery vehicle; and (c) modulating transcription in thesample through the contacting of step (b). Preferably, the nanoparticledelivery vehicle further comprises a tether sequence attached to, anddisposed between, the active agent and the nanoparticle. Preferably, thenanoparticle delivery vehicle further comprises a cell surfacerecognition sequence. Preferably, the nanoparticle delivery vehicle isdisposed in a pharmaceutically acceptable diluent. Preferably, thenanoparticle delivery vehicle further comprises a detectable moiety.

A method of modulating RNA splicing in a sample is disclosed. The methodcomprises: (a) providing a nanoparticle delivery vehicle of the presentinvention comprising a nucleic acid sequence known or suspected to alterthe splicing pattern for a target gene; and (b) contacting a samplecomprising the target gene with the nanoparticle delivery vehicle; and(c) modulating RNA splicing in a sample through the contacting of step(b). Preferably, the nanoparticle delivery vehicle further comprises atether sequence attached to, and disposed between, the active agent andthe nanoparticle. Preferably, the nanoparticle delivery vehicle furthercomprises a cell surface recognition sequence. Preferably, thenanoparticle delivery vehicle is disposed in a pharmaceuticallyacceptable diluent. Preferably, the nanoparticle delivery vehiclefurther comprises a detectable moiety.

A method of modulating the translation of an mRNA sequence encoding aprotein of interest is disclosed. The method comprises: (a) providing ananoparticle delivery vehicle of the present invention comprising asingle stranded nucleic acid sequence complementary to a nucleic acidsequence of an mRNA sequence encoding a protein of interest; (b)contacting a sample comprising the mRNA sequence encoding a protein ofinterest with the nanoparticle delivery vehicle; and (c) modulating thetranslation of an mRNA sequence encoding a protein of interest throughthe contacting of step (b). Preferably, the nanoparticle deliveryvehicle further comprises a tether sequence attached to, and disposedbetween, the active agent and the nanoparticle. Preferably, thenanoparticle delivery vehicle further comprises a cell surfacerecognition sequence. Preferably, the nanoparticle delivery vehicle isdisposed in a pharmaceutically acceptable diluent. Preferably, thenanoparticle delivery vehicle further comprises a detectable moiety.

Accordingly, it is an object of the present invention to provide ananoparticle delivery vehicle. This and other objects are achieved inwhole or in part by the present invention.

Some of the objects of the invention having been stated hereinabove,other objects will be evident as the description proceeds, when taken inconnection with the accompanying Drawings as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a transmission electron micrograph of hepatocytes grown in amedium comprising 5 nm nanoparticles. A region of the nucleus ishighlighted in the inset.

FIG. 1B is a transmission electron micrograph of hepatocytes grown in amedium comprising 30 nm nanoparticles. A region of the cytoplasmincluding a vacuole is highlighted in the inset.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nanoparticle delivery vehicle as avehicle for carrying proteins, nucleic acids, protein and nucleic acidanalogs, small molecules and other compounds to the surface of a cell,into the cytoplasm of a cell and/or into a cell's nucleus. A pluralityof sequences can be associated with a nanoparticle delivery vehicle,preferably a plurality of different sequences, such as RME sequences andNLS sequences. These sequences can aid in the translocation of a vehicleacross various membranes, such as the nuclear membrane of a cell or theouter membrane of a cell. Thus, if membranes and other structures thatgenerally inhibit translocation of a vehicle to a given location in oron a cell are analogized as “locks”, NLS and RME sequences can beanalogized to be “keys”. Thus, in a preferred embodiment, a nanoparticledelivery vehicle of the present invention can comprise a plurality ofdifferent sequences or “keys,” which can enable a given nanoparticledelivery vehicle to pass through various potential barriers totranslocation in a variety of different cell types.

I. Definitions

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

As used herein, the term “active agent” means a therapeutic agent,including but not limited to chemotherapeutic agents, radiotherapeutics,or radiosensitizing agents; an imaging agent; a diagnostic agent; orother agent known to interact with an intracellular protein, a nucleicacid or a soluble or insoluble ligand.

As used herein, the term “amino acid sequence” means an oligopeptide,peptide, polypeptide, or protein sequence, and fragments thereof, andnaturally occurring or synthetic molecules. Where “amino acid sequence”is recited herein to refer to an amino acid sequence of a syntheticpeptide or a naturally occurring protein molecule, amino acid sequence,and the like. The term is not meant to limit the amino acid sequence toa complete, native amino acid sequence associated with a recited proteinmolecule, but is intended to encompass variations on the native aminoacid sequence as well.

As used herein, the term “biodegradable” means any structure, includingbut not limited to a nanoparticle, which decomposes or otherwisedisintegrates after prolonged exposure to physiological conditions. Tobe biodegradable, the structure should be substantially disintegratedwithin a few weeks after introduction into the body. Brushite is apreferred biodegradable nanoparticle material.

As used herein, the terms “extracellular targeting agent” and “cellsurface recognition sequence” are used interchangeably and refer to asmall molecule or protein sequence that is recognized and bound by oneor more receptors present on the surface of a particular cell. Cellsurface recognition sequences can include HIV coat proteins (gp160, 41,120) corona virus coat proteins, EBV coat proteins (gp350) and peptides.Other representative, but non-limiting cell surface recognitionsequences can comprise carbohydrate and lipid, carbohydrates, peptidenucleic acids, morpholino oligonucleotides and polymers. It is intendedthat the term “cell surface recognition sequence” encompass any sequenceor molecule recognized and/or bound by a cell surface receptor. It ispreferable, but not required, that a “cell surface recognition sequence”that is recognized and/or bound by a cell surface receptor leads toreceptor-mediated endocytosis (RME). A list of representative moietiesthat can be employed as targeting agents for internalization by RME ispresented in Table 1.

As used herein, the term “chemical modification” means alteration of afirst moiety by covalently, noncovalently or ionically binding a secondmoiety to the first moiety. Chemical modification can involve theaddition of a detectable moiety to a peptide or protein.

As used herein, the term “detecting” means confirming the presence of atarget entity by observing the occurrence of a detectable signal, suchas an electrical, radiological or spectroscopic signal that will appearexclusively in the presence of the target entity. The term encompassesthe use of electrophoresis techniques and blotting techniques, includingnorthern, Southern, western and far western blots. The term “detecting”also includes the use of microscopy techniques, such as transmissionelectron microscopy. “Detecting” an event or the presence of a compoundcan be done directly or indirectly, for example, by monitoring the rateof transcription to detect the presence of transcription factors. Thus,the term “detecting” broadly means identifying the presence or absenceof an event, compound, molecule, etc.

As used herein, the term “gene” is used for simplicity and means afunctional protein, polypeptide or peptide encoding unit. As will beunderstood by those in the art, this functional term includes bothgenomic sequences and cDNA sequences.

As used herein, the term “gold” means element 79, which has the chemicalsymbol Au; the term specifically excludes any connotation related tocolor or other colorimetric properties.

As used herein, the term “homology” means a degree of complementarity.There can be partial homology or complete homology (i.e., identity). Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid can beconsidered “substantially homologous”. The inhibition of hybridizationof the completely complementary sequence to the target sequence can beexamined using a hybridization assay (Southern or northern blot,solution hybridization and the like) under conditions of low stringency.A substantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of low stringency. This is not to saythat conditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding can be tested by the use of a secondtarget sequence that lacks even a partial degree of complementarity(e.g., less than about 30% identity). In the absence of non-specificbinding, the probe will not hybridize to the second non-complementarytarget sequence.

As used herein, the term “hybridization” means the binding of a probesample to a target sample. The probe sample can comprise a molecule towhich a detectable moiety has been bound, thereby making it possible todetect the presence or absence of a probe sample.

As used herein, the term “interact” means detectable interactionsbetween molecules, such as can be detected using, for example,transmission electron microscopy or fluorescence microscopy. The term“interact” is also meant to include “binding” interactions betweenmolecules. Interactions can be, for example, nucleic acid-nucleic acid,protein-protein or protein-nucleic acid in nature.

As used herein, the term “isolated” means oligonucleotides substantiallyfree of other nucleic acids, proteins, lipids, carbohydrates or othermaterials with which they can be associated, such association beingeither in cellular material or in a synthesis medium. The term can alsobe applied to polypeptides, in which case the polypeptide will besubstantially free of nucleic acids, carbohydrates, lipids and otherundesired polypeptides.

As used herein, the term “labeled” means the covalent, noncovalent orionic attachment of a moiety capable of detection by electrochemical,spectroscopic, radiologic or other methods to a probe molecule.

As used herein, the term “modified” means an alteration from an entity'snormally occurring state. An entity can be modified by removing discretechemical units or by adding discrete chemical units. The term “modified”encompasses detectable labels as well as those entities added as aids inpurification. Any variation from the normally occurring state,regardless of degree, is encompassed by the term “modified”.

As used herein, the term “modulate” means an increase, decrease, orother alteration of any, or all, chemical and biological activities orproperties of a sample which are mediated by a nucleic acid sequence, apeptide or a small molecule. The term “modulation” as used herein refersto both upregulation (i.e., activation or stimulation) anddownregulation (i.e. inhibition or suppression) of a response orproperty.

As used herein, the term “mutation” carries its traditional connotationand means a change, inherited, naturally occurring or introduced, in anucleic acid or polypeptide sequence, and is used in its sense asgenerally known to those of skill in the art.

As used herein, the terms “nano”, “nanoscopic” “nanometer-sized”,“nanostructured”, “nanoscale”, “DNA-nanoparticle complexes” andgrammatical derivatives thereof are used synonymously andinterchangeably and mean nanoparticles, nanoparticle composites andhollow nanocapsules less than or equal to about 1000 nanometers (nm) indiameter, preferably less than about 30 nanometers in diameter and morepreferably less than about 10 nanometers in diameter. A nanoparticle canbe fashioned from any material. A preferred nanoparticle is fashioned ofa semiconductor material or metal, and more preferably of gold, TiO₂ orgold or TiO₂-containing materials. Biodegradable materials are alsopreferred, e.g. polypeptides. The terms can refer not only to the metalcomponent of a nanoparticle, but the composite of metal and othercomponent parts as well.

The term “nanoparticle” as used herein denotes a carrier structure whichis biocompatible with and sufficiently resistant to chemical and/orphysical destruction by the environment of use such that a sufficientamount of the nanoparticles remain substantially intact after injectioninto the blood stream, given intraperitoneally or orally or incubatedwith an in vitro sample so as to be able to reach the nucleus of a cellor some other cellular structure. If the drug can enter the cell in theform whereby it is adsorbed to the nanoparticles, the nanoparticles mustalso remain sufficiently intact to enter the cell. Biodegradation of thenanoparticle is permissible upon entry of a cell's nucleus.Nanoparticles can be solid colloidal particles ranging in size from 1 to1000 nm. Nanoparticle can have any diameter less than or equal to 1000nm, including 5, 10, 15, 20, 25, 30, 50, 100, 500 and 750 nm. Drugs,active agents, bioactive or other relevant materials can be incubatedwith the nanoparticles, and thereby be adsorbed or attached to thenanoparticle.

As used herein, the term “nanoparticle metal component” means acomponent of a nanoparticle delivery vehicle of the present invention towhich a nuclear localization signal, drugs, bioactive and other relevantmaterials are bound. Typically, but not necessarily, the nanoparticlemetal component comprises an approximately spherical metalatom-comprising entity. Preferably the nanoparticle metal component isan elemental metal or semiconductor material, such as a gold or TiO₂particle.

As used herein, the term “nuclear localization signal” means an aminoacid sequence known to, in vivo, direct a protein disposed in thecytoplasm of a cell across the nuclear membrane and into the nucleus ofthe cell. A nuclear localization signal can also target the exteriorsurface of a cell. Thus, a single nuclear localization signal can directthe entity with which it is associated to the exterior of a cell and tothe nucleus of a cell. Such sequences can be of any size andcomposition, for example more than 25, 25, 15, 12, 10, 8, 7, 6, 5 or 4amino acids, but will preferably comprise at least a four to eight aminoacid sequence known to function as a nuclear localization signal (NLS).

As used herein, the term “pharmaceutically acceptable” and grammaticalvariations thereof, as it refers to compositions, carriers, diluents andreagents, means that the materials are capable of administration to orupon a vertebrate subject without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset, feverand the like.

As used herein, the terms “polypeptide”, “protein”, “gene product” and“peptide” are used interchangeably and mean any polymer comprising anyof the 20 protein amino acids, regardless of its size. Although“protein” is often used in reference to relatively large polypeptides,and “peptide” is often used in reference to small polypeptides, usage ofthese terms in the art overlaps and varies. The term “polypeptide” asused herein refers to peptides, polypeptides and proteins, unlessotherwise noted. As used herein, the terms “protein”, “polypeptide” and“peptide” are used interchangeably herein when referring to a geneproduct.

As used herein, the term “sequencing” means determining the orderedlinear sequence of nucleic acids or amino acids of a DNA or peptide (orprotein) target sample, using manual or automated laboratory techniquesknown in the art.

As used herein, the term “small molecule” means a molecule that has amolecular weight of less than or equal to 5000 daltons.

As used herein, the term “substantially pure” means that thepolynucleotide or polypeptide is substantially free of the sequences andmolecules with which it is associated in its natural state, and thosemolecules used in the isolation procedure. The term “substantially free”means that the sample is at least 50%, preferably at least 70%, morepreferably 80% and most preferably 90% free of the materials andcompounds with which is it associated in nature.

As used herein, the term “targeting agent” means any agent having theability to direct a moiety associated with the targeting agent to thesurface of a cell, to the surface of a particular type of cell, or tothe nucleus of a cell. A targeting agent can comprise, but is notlimited to, proteins, peptides, small molecules, oligonucleotides,morpholino oligonucleotides and peptide nucleic acids. A targeting canbe of any size, as long as it retains its ability to direct a moietyassociated with the targeting agent to the surface of a cell or to thesurface of a particular type of cell.

As used herein, the term “therapeutic agent” means any agent having atherapeutic effect, including but not limited to chemotherapeutics,toxins, radiotherapeutics, or radiosensitizing agents. Also encompassedby the term are gene therapy vectors, antisense nucleic acid constructsand transcription factor decoys.

As used herein, the term “transcription factor” means a polypeptide thatis involved in the transcription of DNA. Transcription factors can, butare not required to bind DNA. A transcription factor can function inresponse to an external stimulus, or a transcription factor's action canbe constitutive.

As used herein, the terms “transcription factor decoy” and “decoy” areused interchangeably and mean molecules that bind to or interact withtranscription factors and/or prevent their binding to native enhancersequences. Decoys include nucleic acid sequences, including, but notlimited to, oligonucleotides that correspond to (i.e., are identical toor essentially identical to) the native enhancer. Such oligonucleotidesinclude, but are not limited to: single stranded palindromicoligonucleotides comprising one or more repeats of the enhancersequence; sense and antisense oligonucleotides comprising one or morerepeats of the enhancer sequence; oligonucleotides that form hairpinstructures such that a duplex binding site for the transcription factoris generated; and one or more oligonucleotides that form a cruciformstructure such that one or more binding sites for the transcriptionfactor are generated; and double stranded DNA sequences that have ahigher affinity for a genomic binding site of a transcription factorthan does the natural DNA sequence.

As used herein, the term “wild-type” means the naturally occurring formof a protein or nucleic acid sequence. The term is not used to denote abaseline from which a mutation is established. The term “wild-type” ismeant to describe the form of a protein or nucleic acid sequence as itis most commonly found in nature.

II. General Considerations

The present invention pertains in part to the regulation and modulationof gene expression. Gene expression can be regulated by placing aforeign DNA or RNA oligonucleotide (or an analog such asphosphorothionate DNA/RNA) in the cell for the purpose of (a)incorporation into the genome; (b) expression of a gene (which issometimes considered “transient transfection”); (c) altering regulatoryprotein concentrations (wherein a vehicle can act as a transcriptionfactor decoy); (d) altering RNA splicing; (e) binding to messenger RNAin the cytoplasm, (f) RNA interference or (g) altering the expression ofa segment of DNA by inducing the formation of untranscribablestructures, such as a triple helix. The strategy for (a), (b) and (g)generally involves the delivery of a relatively long double stranded DNAoligomer to the nucleus. The strategy for (c) through (g) can involve ashort DNA oligomer that is an operator (i.e. sequence of DNA known toact as a binding site) for a particular regulatory protein.

The strategy for (d) and (e) can involve RNA, DNA or analogs such aspeptide nucleic acids and morpholino DNA/RNA oligonucleotides as well asthe use of antisense oligonucleotides. However, there has been a greatdeal of difficulty implementing antisense strategies. At the presenttime, it is thought that perhaps this is related to a delivery problemor, alternatively, that the cell has a mechanism for overcoming thereduction in concentration of a particular message. In either case,delivery of oligonucleotides to the nucleus is still a requirement forstrategies (a) through (d).

For strategies (e) and (f), it might only be necessary to deliver anoligonucleotide to the cytoplasm, but this will depend on how theoligonucleotide is to be intercepted. Consequently, the controlleddelivery of oligonucleotides is a key to understanding the mechanism andeffecting control of gene expression. The present invention directlyaddresses this historic antisense problem.

A goal of the present invention is the regulation and/or perturbation ofgene expression. This is of use both for research purposes and also intherapeutic applications. Delivery is a major obstacle in the use ofoligonucleotides and chemically modified oligonucleotides for theseapplications. Nanoparticles of various compositions can be used toachieve a desired result. Materials such as titanium, titanium dioxide,tin, tin oxide, silicon, silicon dioxide, iron, iron^(III) oxide,silver, nickel, gold, copper, aluminum and other materials can be used,however gold is a preferred material. Gold nanoparticles possess severaladvantages. First, gold nanoparticles offer the ability to easilyregulate nanoparticle size and, as explained below, subcellularlocalization. Additionally, synthesis of such nanoparticles is facile,and many art-recognized techniques are available.

Nanoparticles can be conveniently produced by known methods, includingemulsion polymerization in a continuous aqueous phase, emulsionpolymerization in continuous organic phase, interfacial polymerization,solvent deposition, solvent evaporation, dissolution of an organicpolymer solution, cross-linking of water-soluble polymers in emulsion,dissolution of macromolecules, and carbohydrate cross-linking. Thesefabrication methods can be performed with a wide range of materials.Metal atoms, and structures comprising metal atoms, can also serve aseffective nanoparticles. Nanoparticles can be solid or can comprise ahollow structure that can contain a material.

A delivery vehicle of the present invention can comprise one or moreappropriate oligonucleotides associated with a nanoparticle. Next, anuclear localization signal or other localization peptides that willhelp with transport and direct the nanoparticle to the nucleus areassociated with the nanoparticle. The size of the nanoparticle can beused to prevent transport to the nucleus when this is desirable.Finally, appropriate proteins can also be localized on the surface ofthe particle. Appropriate proteins can comprise ligases, restrictionenzymes or other DNA processing enzymes useful for a given application.The localization and effect of the delivery vehicle can then beidentified using transmission electron microscopy, Raman microscopy,confocal microscopy and other analytical techniques for determining theconcentration and localization of the active nanoparticles in the cell.Even delivery vehicles comprising nanoparticles as small as 5 nm can beidentified using one or more of the above analytical techniques.

Thus, the present invention provides a novel approach to solving theproblems of nanoparticles as drug delivery vehicles encountered in theart. Specifically, the present invention discloses nanoparticles that donot necessarily encapsulate a biologically active structure, but ratherserve as a scaffold for the biologically active structure to be attachedto the surface of the nanoparticle. Significantly, the nanoparticles ofthe present invention can also comprise a nuclear localization signal,which can target a therapeutic agent to the nucleus of a cell. Until thedisclosure of the present invention, nuclear localization signals havenot been used to direct a nanoparticle across both the plasma membraneand the nuclear membrane.

In another aspect of the present invention, a plurality of sequences canbe associated with a nanoparticle delivery vehicle. Various sequences,such as RME sequences, can also be associated with a vehicle. Theseadditional sequences can aid in the translocation of a vehicle acrossvarious membranes, such as the nuclear membrane of a cell or the outermembrane of a cell. Thus, if membranes and other structures thatgenerally inhibit translocation of a vehicle to a given location in oron a cell are analogized as “locks”, NLS and RME sequences can beanalogized to be “keys”. Thus, in a preferred embodiment, a nanoparticledelivery vehicle of the present invention can comprise a plurality ofdifferent sequences or “keys,” which can enable a given nanoparticledelivery vehicle to pass through various potential barriers totranslocation and can provide for the targeting of a variety ofdifferent cell types.

The present invention describes a nanoparticle delivery vehicle that canbe used with a variety of subjects including warm blooded animals,particularly mammals, including humans, dogs, cats and other smallanimals, and farm animals. Additionally, the nanoparticles of thepresent invention can be used with prokaryotic and eukaryoticmicroorganisms and with in vitro cultures. The nanoparticle deliveryvehicle of the present invention can be used as a diagnostic agent inall of the above subjects, as well as in the capacity of a therapeuticagent. There is no limitation on the type of biologically activestructure the subject to which a nanoparticle of the present inventioncan be introduced. See, e.g., U.S. Pat. Nos. 5,783,263 and 6,106,798.See also, Colloidal Drug Delivery Systems, (1994) (Kreuter, ed.), MarcelDekker, Inc., New York, pp 219-342; Kreuter, (1994) Eur. J. Drug Metab.Ph. 3: 253-56.

IV. Selection and Preparation of a Targetable Nanoparticle DeliveryVehicle

A targetable nanoparticle delivery vehicle of the present inventionpreferably comprises at least three components: a nanoparticle, one ormore targeting agents (e.g. “keys” such as nuclear localization signalsand cell surface targeting signals) and one or more active agents. Theactive agent can be one or more of any chemical entity, for example, apeptide sequence, a single stranded nucleic acid oligomer, a doublestranded nucleic acid oligomer, a peptide nucleic acid or a smallmolecule. These three components are prepared and joined together tofunction as a delivery vehicle, which can be targeted a cell's nucleusvia the nuclear localization signal. Active agents and nuclearlocalization signals can be synthesized using primers or templatespreviously associated with the nanoparticle. The nuclear localizationsignal directs the translocation of the delivery vehicle to the nucleusof a cell, whereupon the active agent can interact with one or moreproteins or nucleic acids to facilitate a desired effect. If ananoparticle of larger size is selected, for example greater than orequal to about 30 nm, the delivery vehicle will be translocated to acell's cytoplasm.

A targetable nanoparticle delivery vehicle of the present invention canfurther comprise an extracellular targeting agent. In this embodiment, ananoparticle delivery vehicle can comprise two targeting signals. First,a targeting agent can be selected which can direct a delivery vehicle tothe surface of a target structure that recognizes the selected targetingagent. Second, a nuclear localization sequence can be included, whichwill direct a delivery vehicle to the nucleus of a target structure.

IV.A. Selection and Preparation of a Nanoparticle

There are no limits on the physical parameters of a nanoparticlecomponent of the present invention, although the design of a deliveryvehicle should take into account the biocompatibility of thenanoparticle vehicle, where appropriate. The physical parameters of ananoparticle vehicle can be optimized, with the desired effect governingthe choice of size, shape and material. Preferred particle sizes fortransport to a cell's nucleus are on the order of 5 nm although, asdiscussed below, larger particles might be desired for a givenapplication. Additionally, particles smaller than about 25 nm indiameter are preferred for use in nuclear targeting to facilitate entryinto the nucleus via a nuclear pore. (Feldherr & Akin, (1990) ElectronMicrosc. Rev. 3(1):73-86; Feldherr et al., (1992) Proc. Natl. Acad. Sci.U.S.A. 89:11002-5; Feldherr & Akin, (1999) J. Cell Sci. 112:2043-48;Feldherr & Akin, (1994) Exp. Cell Res. 215:206-10.)

The nanoparticle, which can also be referred to as a scaffold, of ananoparticle delivery vehicle can comprise a variety of inorganicmaterials including, but not limited to, metals, semi-conductormaterials or ceramics. Preferred metal-based compounds for themanufacture of nanoparticles include titanium, titanium dioxide, tin,tin oxide, silicon, silicon dioxide, iron, iron^(lll) oxide, silver,gold, copper, nickel, aluminum, steel, cobalt-chrome alloys, cadmium(preferably cadmium selenide) and titanium alloys. Preferred ceramicmaterials include brushite, tricalcium phosphate, alumina, silica, andzirconia. The nanoparticle can be made from organic materials includingcarbon (diamond). Preferred polymers include polystyrene, siliconerubber, polycarbonate, polyurethanes, polypropylenes,polymethylmethacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene. Biodegradable, biopolymer (e.g. polypeptides such as BSA,polysaccharides, etc.), other biological materials (e.g. carbohydrates),and/or polymeric compounds are also suitable for use as a nanoparticlescaffold. Gold is especially preferred due to its well-known reactivityprofiles and biological inertness.

Nanoparticles comprising the above materials and having diameters lessthan 1,000 nanometers are available commercially or they can be producedfrom progressive nucleation in solution (e.g., by colloid reaction), orby various physical and chemical vapor deposition processes, such assputter deposition. See, e.g., Hayashi, (1987) Vac. Sci. Technol.July/August 1987, A5(4):1375-84; Hayashi, (1987) Physics Today, December1987, pp. 44-60; MRS Bulletin, January 1990, pgs. 16-47.

Alternatively, nanoparticles can be produced using HAuCl₄ and acitrate-reducing agent, using methods known in the art. See, e.g.,Marinakos et al., (1999) Adv. Mater. 11: 34-37; Marinakos et al., (1998)Chem. Mater. 10: 1214-19; Enustun & Turkevich, (1963) J. Am. Chem. Soc.85: 3317. Tin oxide nanoparticles having a dispersed (in H₂O) aggregateparticle size of about 140 nm are available commercially from VacuumMetallurgical Co., Ltd. of Chiba, Japan. Other commercially availablenanoparticles of various compositions and size ranges are available, forexample, from Vector Laboratories, Inc. of Burlingame, Calif.Biodegradable, ceramic and polymeric nanoparticle materials will beknown to those of skill in the art and can comprise a biodegradablecomposition.

Besides sputter deposition, plasma-assisted chemical vapor deposition(PACVD) is another technique that can be used to prepare suitablenanoparticles. PACVD functions in relatively high atmospheric pressures(on the order of one torr and greater) and is useful for generatingparticles having diameters of about 1000 nanometers and smaller. Forexample, aluminum nitride particles having diameters of less than 1000nanometer can be synthesized by PACVD using Al(CH₃)₃ and NH₃ asreactants. The PACVD system typically includes a horizontally mountedquartz tube with associated pumping and gas feed systems. A susceptor islocated at the center of the quartz tube and heated using a 60 KHz radiofrequency source. The synthesized aluminum nitride particles arecollected on the walls of the quartz tube. Nitrogen gas is commonly usedas the carrier of the Al(CH₃)₃. The ratio of Al(CH₃)₃:NH₃ in thereaction chamber is controlled by varying the flow rates of theN₂/Al(CH₃)₃ and NH₃ gas into the chamber. A constant pressure in thereaction chamber of 10 torr is generally maintained to providedeposition and formation of the ultrafine aluminum nitridenanoparticles. PACVD can be used to prepare a variety of other suitablebiodegradable nanoparticles.

The size of a nanoparticle can be an important consideration. Largernanoparticles, on the order of greater than or equal to about 30 nm, areseen to enter cells, but are not translocated across the nuclearmembrane into the nucleus of a cell, as seen in FIG. 1B. Presumably,this effect is directly related to the size of the nanoparticle, since 5nm nanoparticles do cross the nuclear membrane and are translocated intothe nucleus, as seen in FIG. 1A. Thus, selection of the appropriatedelivery vehicle size will be important, but also offers an additionallevel of targetability and facilitates the design and employment ofnanoparticles carrying active agents that need to be located in thecytoplasm and not the nucleus.

IV.B. Selection and Preparation of an Active Agent

Having selected and prepared a nanoparticle to which at least a nuclearlocalization signal and preferably another, different targeting agentare attached, a desired active agent is selected and prepared.Appropriate active agents can comprise any small molecule, protein ornucleic acid sequence, and the selection is governed by the intendedapplication of the delivery vehicle. Various applications ofnanoparticle delivery vehicles of the present invention are discussed indepth below, and include gene therapy, modulation of gene expression,altering RNA splicing and modulation of protein-protein interactions.Appropriate active agents will be apparent to one of skill in the artupon review of the present disclosure and will be selected with toregard to a desired experimental or clinical goal.

It is also an aspect of the present invention to provide a nanoparticledelivery vehicle comprising two or more different active agents, e.g. 2,3, 4, 5, or other desired number of different active agents. Thus,delivery of the different active agents can be accomplished in the samecellular or tissue location, or in two or more different cellular ortissue locations, depending on the targeting sequences that areemployed.

Any combination of any of the active agents disclosed herein can beprovided. For example, a nanoparticle delivery vehicle comprising atherapeutic agent and an imaging agent can be provided, for use in forexample, delivery to a tumor. As another example, a nanoparticledelivery vehicle comprising a chemotherapeutic agent and aradiosensitizing agent can be provided. As yet another example, ananoparticle delivery vehicle comprising different polynucleotidesequences for use in modulation of transcription and/or translation ofthe same or different genes can be provided.

IV.B.1. Selection of an Active Agent

Generally, a single stranded nucleic acid sequence appropriate for useas an active agent in the present invention can be selected on the basisof the context in which the present invention is employed. In oneembodiment, appropriate single stranded DNA are complementary to anucleic acid sequence known or suspected to be present in a diseasecondition. In another embodiment, appropriate single stranded DNA iscomplementary to an overexpressed gene. Functional equivalents of knownsequences can also be used as active agents and are considered to be anaspect of the present invention. Nucleic acid sequences of anymanageable length can be used as an active agent. Typically, such agentsrange between about 20 and about 50 nucleotides in length, althoughlonger sequences can be used. In yet another embodiment, a nucleic acidsequence corresponding to a full-length gene, or a fragment thereof, canbe used as an active agent.

Double stranded DNA of various lengths and compositions is suitable foruse as an active agent in the present invention. Double stranded DNA canbe of any length, from a few base pairs up to the length of afull-length gene. As discussed below, long lengths of DNA, and notablyfull-length genes, find utility in gene therapy applications. In thisembodiment, full-length genes can be incorporated into a host cell'sgenome, or can be transiently expressed within the cell. In thisembodiment, then, a cell is lacking a particular gene and an appropriatedouble stranded DNA sequence selected as an active agent is the geneabsent from the cell's genome.

Nucleic acid analogs can also be used as active agents in the presentinvention. In one aspect of the present invention, peptide nucleic acidanalogs (PNAs) can be used as active agents. A peptide nucleic acidanalog is a DNA analog wherein the backbone of the analog, normally asugar backbone in DNA, is a pseudopeptide. A PNA backbone can comprise asequence of repeated N-(2-amino-ethyl)-glycine units. Peptide nucleicacid analogs react as DNA would react in a given environment, and canadditionally bind complementary nucleic acid sequences. Peptide nucleicacid analogs offer the potential advantage over unmodified DNA of theformation of stronger bonds, due to the neutrally charged peptidebackbone of the analogs, and can impart a higher degree of specificitythan is achievable by unmodified DNA.

PNAs have been employed in a wide array of biochemical roles, which isapplicable to the present invention, including sequence mapping. Invitro studies indicate that PNA could inhibit both transcription andtranslation of genes to which it has been engineered with acomplementary sequence. This suggests that PNAs could be useful inantigene and antisense therapy. See, e.g., Norden et al., (2000) FASEBJ. 14(9): 1041-60. To date, however, researchers have been unable toreproducibly target such a sequence to the cell nucleus from outside theplasma membrane.

The present invention addresses this problem and offers potential forheretofore unattainable applications of PNAs. PNAs suitable for use asactive agents in the present invention will, therefore, comprise asequence complementary to a sequence of interest. Other nucleic acidanalogs useful in the context of the present invention includemorpholino nucleic acid analogs. Morpholino analogs can be substitutedfor a nucleic acid sequence and has the benefits of both complementarityand a unique chemical reaction and binding profile not found in nativenucleic acid sequences. See, e.g., Chakhmakhcheva et al., (1999)Nucleos. Nucleot. 18: 1427-28.

Additionally, proteins are appropriate for use as an active agent in thepresent invention. In one embodiment, appropriate proteins compriseproteins known to interact with proteins associated with DNA replicationand expression, for example, ligases. In this embodiment, ananoparticle-bound protein can be a protein that interacts with DNA orRNA sequences, possibly as an up- or downregulator of the transcriptionprocess, the translation process or both. In an alternative embodiment,the present invention can be used to prove or disprove a putativeprotein-protein interaction. In this case, the nanoparticle-boundsequence is a probe protein and the application of the invention cangive data similar to that achievable using the well-characterized yeasttwo-hybrid system and other analytical systems, although the presentinvention affords the opportunity to examine such an interaction insitu. More specifically, proteins capable of interacting with receptorson nuclear regulatory proteins can be employed as active agents.

Finally, small molecules attached to a nanoparticle can be used asactive agents in the present invention. Appropriate small molecules willhave the ability to interact with enzymes, cofactors, nucleic acids andother intracellular structures. Small molecules can be those identifiedas natural ligands, inhibitors (competitive, uncompetitive andnoncompetitive) or designed modulators. Chemotherapeutic agents,radiotherapeutics, or radiosensitizing agents; an imaging agent; adiagnostic agent; or other agent known to interact with an intracellularprotein, a nucleic acid or a soluble or insoluble ligand can also beused as active agents.

It should be noted that the use of one of the above active agents doesnot preclude the binding of a different active agent to the nanoparticleand several active agents can be joined to a single nanoparticle.Moreover, active agents can be multivalent and/or multifunctional.

IV.B.2. Preparation of an Active Agent

Nucleic acid sequences useful as active agents in the context of thepresent invention can be prepared in a variety of ways and will beapparent to one of skill in the art upon review of the presentdisclosure. For example, an appropriate DNA sequence can be excised froma larger DNA sample using restriction endonucleases, which sever nucleicacid sequences at known sequences. Excised nucleic acid sequences can beexcised and purified using methods known in the art. See, e.g., Sambrooket al., (1992) Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y. for a general discussion of cloning strategies.Alternatively, and more preferably, nucleic acid sequences can besynthesized using well-known manual and automated nucleic acid synthesismethods. All nucleic acid sequences that are used as active agents,whether they are excised, synthesized or otherwise prepared, should besubstantially pure. Synthesis of nucleic acid or protein active agentscan proceed using a template previously associated with a nanoparticle.

Isolation and purification of proteins will correspond with techniquesestablished for preparation of a given protein; those proteins ofinterest that have not been purified can be isolated using methods knownto those of skill in the art and are not discussed here. Similarly,strategies of synthesizing and purifying small molecules can be found inthe art and will be evident to one of skill in the art of organicchemistry or other chemical discipline.

IV.C. Selection and Preparation of a Nuclear Localization Signal

The inclusion of a nuclear localization signal (NLS) as a deliveryvehicle component is an aspect of the present invention. Arepresentative nuclear localization signal is a peptide sequence thatdirects the protein to the nucleus of the cell in which the sequence isexpressed. A nuclear localization signal is predominantly basic, can bepositioned almost anywhere in a protein's amino acid sequence, generallycomprises a short sequence of four amino acids (Autieri & Agrawal,(1998) J. Biol. Chem. 273: 14731-37) to eight amino acids, and istypically rich in lysine and arginine residues (Magin et al., (2000)Virology 274: 11-16). Nuclear localization signals often compriseproline residues. A variety of nuclear localization signals have beenidentified and have been used to effect transport of biologicalmolecules from the cytoplasm to the nucleus of a cell. See, e.g.,Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:7442-46; Moedeet al., (1999) FEBS Leff. 461:229-34. Translocation is currently thoughtto involve nuclear pore proteins.

In a preferred embodiment of the present invention, a nuclearlocalization signal be attached to the nanoparticle. The nuclearlocalization signal can be synthesized or excised from a largersequence. As noted, a variety of nuclear localization signals are knownand selection of an appropriate sequence can be made based on the knownproperties of these various sequences. Representative NLS's include themonopartite sequence PKKKRKV (SEQ ID NO: 1) and the bipartite sequenceKRPAAIKKAGQAKKKK (SEQ ID NO: 2).

Nuclear localization signals appear at various points in the amino acidsequences of proteins. NLS's have been identified at the N-terminus, theC-terminus and in the central region of proteins. Thus, a selectedsequence can serve as the functional component of a longer peptidesequence. The residues of a longer sequence that do not function ascomponent NLS residues should be selected so as not to interfere, forexample tonically or sterically, with the nuclear localization signalitself. Therefore, although there are no strict limits on thecomposition of an NLS-comprising sequence, in practice, such a sequencecan be functionally limited in length and composition.

In another aspect of the present invention, a plurality of sequences canbe associated with a nanoparticle delivery vehicle. Various sequences,such as RME sequences, can also be associated with a vehicle. Theseadditional sequences can aid in the translocation of a vehicle acrossvarious membranes, such as the nuclear membrane of a cell or the outermembrane of a cell. Thus, if membranes and other structures thatgenerally inhibit translocation of a vehicle to a given location in oron a cell are analogized as “locks”, NLS and RME sequences can beanalogized to be “keys”. Thus, in a preferred embodiment, a nanoparticledelivery vehicle of the present invention can comprise a plurality ofdifferent sequences or “keys,” which can enable a given nanoparticledelivery vehicle to pass through various potential barriers totranslocation.

IV.D. Selection and Preparation of a Cell Surface Receptor RecognitionMoiety

In one aspect of the present invention, a moiety that imparts theability to be recognized and/or bound by a cell surface receptor isbound to the nanoparticle. This moiety will generally comprise a proteinsequence known to be recognized by a cell surface receptor. Preferably,the cell surface receptor recognition moiety can further comprise anucleic acid sequence, either alone or as part of a nucleic acid-proteinhybrid, or peptide analog. A vast number of cell surface receptors areknown and can be useful in the present invention, including themacrophage mannose receptor and its various homologs, and thoseassociated with retroviruses such as HIV.

A representative, but non-limiting, list of moieties that can beemployed as targeting agents in the present invention is presented inTable 1. Homologs of the presented moieties can also be employed. Thesetargeting agents can be associated with a nanoparticle and used todirect the nanoparticle to a target structure, where it can subsequentlybe internalized. There is no requirement that the entire moiety be usedas a targeting agent. Smaller fragments of these moieties known tointeract with a specific receptor or other structure can also be used asa targeting agent. The targeting agents of Table 1 can function tointernalize (e.g., by receptor mediated endocytosis) a delivery agentinteracting with a targeting agent.

TABLE 1 Diptheria Toxin Pseudomonas toxin Cholera toxin RicinConcanavalin A Rous sarcoma virus Semliki forest virus Vesicularstomatitis virus Adenovirus Transferrin Low Density LipoproteinTranscobalamin Yolk Proteins IgE Polymeric IgA Maternal IgG IgG, via Fcreceptors Insulin Epidermal Growth Factor Growth Hormone ThyroidStimulating Hormone Nerve Growth Factor Calcitonin Glucagon ProlactinLuteinizing Hormone Thyroid hormone Platelet Derived Growth FactorInterferon Catecholamines Nuclear Localization Signal

The recognition moiety can further comprise a sequence that is subjectto enzymatic or electrochemical cleavage. The recognition moiety canthus comprise a sequence that is susceptible to cleavage by enzymespresent at various locations inside a cell, such as proteases orrestriction endonucleases (e.g. DNAse or RNAse).

It must be emphasized that a cell surface recognition sequence is not anabsolute requirement for the present invention. Indeed, as shown in FIG.1A, hepatocytes grown in media containing nanoparticles lacking a cellsurface recognition sequence were translocated across the cell membrane,in the absence of such a sequence. Thus, although a cell surfacereceptor sequence can be useful for targeting a given cell type, or forinducing the association of a nanoparticle with a cell surface, there isno requirement that a cell surface recognition sequence be present onthe surface of a nanoparticle in order to practice the presentinvention.

The presence of a cell surface receptor unique to a given type of cellcan assist in the selection and delivery of an active agent to that celltype. For example, macrophages express a cell surface receptor (themacrophage mannose receptor) that mediates pinocytosis of particles thatcomprise mannose through carbohydrate recognition domains. See, Mullinet al., (1997) J. Biol. Chem. 272: 5668-81. Thus, the selection of anappropriate cell surface receptor can facilitate cell specific selectionby a nanoparticle delivery vehicle and consequently, cell specificinteraction.

IV.E. Selection and Preparation of a Tether Sequence

In another aspect of the present invention, a short tether sequence canbe disposed between the nanoparticle and a cell surface recognitionsequence of a nanoparticle delivery vehicle of the present invention.The tether can be a protein sequence, a nucleic acid sequence or anyother composition that is compatible with an intracellular environment.In the present invention, protein and nucleic acid sequences arepreferred due to their enzymatic cleavability. For example, a nucleicacid tether that comprises a known cut site for a restrictionendonuclease found in the targeted cell can be employed. Alternatively,a protein tether can be employed that comprises a cut site for aprotease commonly found in the targeted cell type. Finally, a tether canbe designed that can be chemically or electrochemically cleaved.

Cleavage of a tether is one method by which the nanoparticle, which willcomprise an active agent and a nuclear localization signal, can be freedto translocate across the nuclear membrane and into the nucleus of acell. In one example, upon association of a cell surface receptor with acell surface recognition sequence disposed on the surface of ananoparticle of a delivery vehicle of the present invention, thenanoparticle delivery vehicle is, in effect, bound to the cell surface.Upon translocation of the delivery vehicle to the interior of a cell, byendocytosis or other mechanism, the nanoparticle delivery vehicleremains bound to the receptor. In order to free the nanoparticledelivery vehicle and its associated active agent, the tether can becleave by endogenous proteases, nucleases or other chemical orelectrochemical techniques. Cleavage of the tether sequence frees thenanoparticle delivery vehicle and enables subsequent direction of thedelivery vehicle to the nucleus, via the nuclear localization signaldisposed on the nanoparticle surface.

IV.F. Assembly of a Nanoiarticle Delivery Vehicle

Having selected and prepared the various individual components of ananoparticle delivery vehicle of the present invention, the agent itselfis then assembled. The order of assembly is not critical and can begoverned primarily by the requirements of a desired chemical reaction.The chemical properties of an active agent, an NLS, a nanoparticle, aswell as physiological and various other considerations, should also beweighed. Thus, the assembly procedure described hereinbelow is presentedin an arbitrary order. Further, the materials described, i.e.composition of the nanoparticle, etc., are presented only as examplesand are not meant to be limiting in any way. Suitable materials andsequences will be known to those of skill in the art when evaluated inlight of the present disclosure and the knowledge and resourcesavailable to researchers in the applicable fields.

IV.F.1. Association of an Active Agent with a Nanoparticle

Following selection and preparation of a nanoparticle and a suitableactive agent, the two components are joined to form a complex. In oneembodiment, the nanoparticle is fashioned of gold. Gold is particularlyuseful in the present invention due to its well-known reactivity profileand its relatively inertness in the context of biological systems.Colloidal gold can be used, although the overall negative charge ofcommon preparations of gold imparts the quality that colloidal gold hasa high non-specific affinity for certain proteins. The negative chargeof a preparation can be imparted by association of gold molecules withnegatively-charged ligands such as citrate orbis-sulfonatotriphenylphosphine. These negatively-charged ligands, andthus the overall negative charge associated with a colloidal goldpreparation, are preferably reduced or eliminated by exchanging anynegatively-charged ligands with neutral ligands, such as polyethyleneglycol, or positively-charged ligands, such as amines.

Alternatively, the use of colloidal gold can be accompanied byadditional treatments such that it can be coated at some point in thepreparation process with another protein, such as BSA, in order to blockundesired nonspecific protein binding. Small molecule and peptidecoatings can also be used to avoid specific interactions with cellularproteins. For example, colloidal gold has been prepared with a coatingof glutathione. Since glutathione, a natural antioxidant, is one of themost abundant peptides in the cell, this coating may impart to thenanoparticle a camouflage-like effect. Alternatively, some gold clustersand particles are commercially available and can be used in the presentinvention. For example, NANOGOLD® gold particles are available fromNanoprobes, Inc., Yaphank, N.Y. Also preferable for use in the presentinvention are biodegradable particles, which can be fashioned of anappropriate polymer or other material. Some commercially availablenanoparticles are prepared for labeling prior to shipping and areconvenient for attaching entities to the nanoparticles.

In one embodiment, using a gold nanoparticle as the nanoparticle and asingle or double stranded nucleic acid as an active agent, a thiolationreaction can be performed to add a thiol group to the 5′ end of thenucleic acid oligomer. Alternatively, an amination reaction can beperformed and will proceed mutatis mutandis to the thiolation reactiondescribed herein. The general purpose of the reaction is to introduce anucleophilic center, which can subsequently be functionalized with adesired moiety. A representative thiol modifier phosphoramidite reagentis presented as Compound 1, which is available from Glen Research, Inc.of Sterling, Va.

Nucleic acid oligmers are incubated with a thiol modifierphosphoramidite under conditions that permit attachment of the phosphineto the 5′ end of the probe DNA. The reaction can be carried out in a DNAsynthesizer using standard conditions. Compound 1 can be added as a stepin automated DNA synthesis using an automated apparatus, such as theABI® 3900 high-throughput DNA synthesizer (Applied Biosystems, FosterCity, Calif.). The thiol modifier is added in the last step of synthesisof an oligonucleotide. The phosphine is oxidized using iodine and thepurification is exactly the same as that used for unlabeledoligonucleotides. The purification process is easier for labeledoligonucleotides since labeled oligonucleotides are significantly morehydrophobic and therefore tend to elute much more slowly under typicalHPLC conditions. The phosphoramidite reacts spontaneously with the 5′hydroxyl of DNA, which can be disposed in acetonitrile. In thisreaction, the thiol group is protected by a protecting trityl or aceticthioester group and is separated from the 5′-phosphodiester by avariable length carbon linker. A six-carbon linker is present inCompound 1.

The nucleic acid complex is then subjected to thiol deprotection toremove the trityl group. Specifically, the protecting trityl group isremoved by treatment with silver nitrate and dithiothreitol (DTT). Thenucleic acid complex is incubated with a nanoparticle metal component.The two entities are joined at the thiol exposed by the removal of thetrityl group during the deprotection reaction. The formed activeagent-nanoparticle complexes (in this embodiment nucleicacid-nanoparticle complexes) can be maintained in the reaction vesseluntil use.

IV.F.2. Association of a Nuclear Localization Signal with a Nanoparticle

A suitable nuclear localization signal is joined to the activeagent-nanoparticle complex. Nuclear localization signals can besynthesized using standard peptide chemistry techniques, or can beisolated by proteolytic cleavage from a larger protein. Isolated orsynthesized nuclear localization signals can be of any size, with theonly requirement that the sequence comprise at least a known NLS, whichare typically four to eight amino acids in length. Protein and peptidepurification methods suitable for preparing nuclear localizationsignals, which are isolated from larger proteins, are known in the art.See generally, Protein Purification Applications: A Practical Approach,(1989) (Harris & Angal, eds.) IRL Press; Protein Purification:Principles, High Resolution Methods, Applications, (1989) (Janson &Ryden, eds.) VCH Publishers.

The present invention also encompasses the preparation of andassociation of a protein-peptide conjugate with a nanoparticle. Such aconjugate can comprise, for example, a comparatively large protein and acomparatively small NLS. Although both entities comprise amino acids,the distinction of protein and peptide is made based on, among othercriteria, the functionality of each entity. In a specific example, aprotein-peptide conjugate can comprise BSA and an NLS. Protein-peptideconjugates can subsequently be bound to gold nanoparticles.

The chemistry of attaching proteins and peptides to gold nanoparticlesis similar to the chemistry required for attaching nucleic acids to goldnanoparticles. In one aspect, a thiol reaction is performed. Thereaction can involve a thiol group disposed on the nuclear localizationsignal, which can take the form of a terminal cysteine or methionineresidue, or on the nanoparticle. The thiol group can be convenientreacted with a primary amine on the alternate entity. The primary aminecan conveniently take the form of a terminal lysine or arginine residuein the nuclear localization signal, but can also be disposed on thesurface of the nanoparticle. See, e.g., Hainfeld & Furuva, (1992) J.Histochem. Cytochem. 40: 177-84; Hainfeld, (1992) Ultramicroscopy 46:135-44.

IV.F.3. Association of a Cell Surface Recognition Sequence with aNanoparticle

An appropriate cell surface recognition sequence can be selected (forexample, one selected from or based on those presented in Table 1) andprepared as described above in Section IV.D above. The sequence can be,essentially, a protein or a peptide known to bind to a receptorexpressed on the surface of a given cell type. Thus, the same chemicalreactions can be performed to associate a cell surface recognitionsequence with a nanoparticle as are performed to associate a nuclearlocalization signal or a protein active agent with a nanoparticle.Continuing with the example from Section IV.F.2 above, a thiol-aminereaction can be performed to associate a thiol disposed on either thenanoparticle or the cell surface recognition sequence with a primaryamine disposed on the alternate member of a thiol-amine reaction pair.Depending on the reactivity profiles and the order in which the variouscomponent parts of a nanoparticle delivery vehicle are bound to ananoparticle, a scheme of site blocking can be developed. Such a schemecan prevent binding of an entity to undesired sites on the nanoparticleor on the component parts themselves.

IV.F.4. Association of a Tether Sequence with a Nanoparticle

A tether sequence can also be bound to a nanoparticle. Such a sequencecan be disposed between the nanoparticle and a cell surface recognitionsequence or other entity associated with the nanoparticle. In order toserve its purpose, the tether sequence preferably comprises a site atwhich chemical, electrochemical or enzymatic cleavage can take place.When a tether sequence comprises a single or double stranded nucleicacid sequence, the sequence can comprise a cut site for a nuclease knownto be present in the cells to which the delivery vehicle is beingintroduced. When the tether is a protein, it can comprise a proteolyticsite. Finally, when it is desired that the tether be cleavedphotolytically, it can comprise a material amenable to photocleavage.Commercially available photocleavable tether sequences include variousspacer phosphoramidites, available from Glen Research of Sterling, Va.

IV.F.5. Biocompatibility and Protection of the Delivery Vehicle

If the nanoparticle comprises a metal component such as gold, it isdesirable to assure biocompatibility between the nanoparticle and asubject to which the delivery vehicle is being administered. Gold isrelatively inert and less physiologically intrusive than other metals,but the detrimental effects of any metal or polymer nanoparticlematerial can be minimized by coating or otherwise wholly or partlycovering the nanoparticle with a biocompatible substance. Compounds thatcan be used to achieve biocompatibility include polymers (such aspolyethylene glycol-PEG), proteins (such as BSA), lipids (includingmembrane envelopes) and carbohydrates. Addition of thesebiocompatibility compounds can be performed following the addition ofthe other delivery vehicle components and can serve as the finalsynthetic step before introduction of the delivery vehicle to a subjector system.

These materials can also protective or masking agents for the deliveryvehicle and the active agent(s) and targeting agent(s) attached theretoto prevent recognition by the immune system or other biological systems(e.g. proteases, nucleases (e.g. DNAse or RNAse), or other enzymes orbiological entities associated with undesired degradation). Thus, theprotective coating or shell provides cloaking or stealth features tofacilitate that the delivery vehicle reaches a desired cell or tissuewith the active agent(s) and targeting agent(s) intact.

IV.F.6. Associating Multiple Sequences with a Delivery Vehicle

Multiple sequences can be associated with a delivery vehicle of thepresent invention. By associating multiple sequences with a singlevehicle, the vehicle can be adapted to pass through various cellularbarriers, such as the cell membrane or a nuclear membrane. For example,a nanoparticle vehicle can comprise an NLS and a RME sequence. The RMEsequence can assist in the translocation of a vehicle across themembrane of a cell. Once inside a cell, the NLS can target the vehicleto the nucleus of the cell.

Preferably any sequences associated with a nanoparticle delivery vehicleare independently associated with the vehicle, rather than formingcomponents of a single long sequence. As indicated by the resultsdisclosed in Laboratory Example 1, independent association of multiplesequences (preferably multiple different sequences) is a more efficientmethod targeting a nanoparticle delivery vehicle to a desired cellularstructure. However, sequential association of multiple sequences canalso be an effective method of directing a nanoparticle delivery vehicleto a given site, and this approach forms another aspect of the presentinvention.

V. Introduction of a Nanoparticle Delivery Vehicle to a Subject orSample

After a sufficiently pure nanoparticle delivery vehicle (preferablycomprising a nanoparticle, an active agent and an NLS) has beenprepared, it might be desirable to prepare the vehicle in apharmaceutical composition that can be administered to a subject orsample. Preferred administration techniques include parenteraladministration, intravenous administration and infusion directly intoany desired target tissue, including but not limited to a solid tumor orother neoplastic tissue. This can be achieved by employing a finalpurification step, which disposes the vehicle in a medium comprising asuitable pharmaceutical composition.

Suitable pharmaceutical compositions in accordance with the inventiongenerally comprise an amount of the desired delivery vehicle-activeagent in accordance with the dosage information (which is determined ona case-by-case basis), admixed with an acceptable pharmaceutical diluentor excipient, such as a sterile aqueous solution, to give an appropriatefinal concentration in accordance with the dosage information set forthabove with respect to the active agent. Such formulations will typicallyinclude buffers such as phosphate buffered saline (PBS), or additionaladditives such as pharmaceutical excipients, stabilizing agents such asBSA or HSA, or salts such as sodium chloride.

For parenteral administration it is generally desirable to furtherrender such compositions pharmaceutically acceptable by insuring theirsterility, non-immunogenicity and non-pyrogenicity. Such techniques aregenerally well known in the art as exemplified by Remington'sPharmaceutical Sciences, (1980) (Osol, ed.) 16th Ed., Mack PublishingCompany, Easton, Pa., incorporated herein by reference. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiological Standards.

When delivery vehicles are being introduced into cells suspended in acell culture, it is sufficient to incubate the cells together with thenanoparticle delivery vehicles an appropriate growth media, for exampleLuria broth (LB) or a suitable cell culture medium. Although otherintroduction methods are possible, these introduction treatments arepreferable and can be performed without regard for the entities presenton the surface of a delivery vehicle.

When in vitro experiments are to be performed, delivery vehicles can beadded to directly to a selected cell growth medium before cells areintroduced into the medium. Such a medium must, obviously, be compatiblenot only with the physiological requirements of the cells, but also withthe chemical and reactivity profile of the delivery vehicle. Thedelivery vehicle's profile will be apparent to one of skill in the artupon review of the present disclosure and in view of the moieties boundto the nanoparticle.

V.A. Receptor Mediated Endocytosis of a Delivery Vehicle

Recognition and binding of a cell surface recognition sequence disposedon a nanoparticle delivery vehicle of the present invention is an aspectof the present invention. The present invention takes advantage of theunderstanding that a cell surface binding event is often the initiatingstep in a cellular cascade leading to a range of events, notablyreceptor-mediated endocytosis.

The above methods describe methods by which a delivery vehicle can beintroduced into a sample or subject. These agents are translocatedacross the cell membrane in a variety of ways. However, when a cellrecognition sequence is bound to a nanoparticle, a different type ofinternalization can occur, namely receptor mediated endocytosis.

The term “receptor mediated endocytosis” (“RME”) generally describes amechanism by which, catalyzed by the binding of a ligand to a receptordisposed on the surface of a cell, a receptor-bound ligand isinternalized within a cell. Many proteins and other structures entercells via receptor mediated endocytosis, including insulin, epidermalgrowth factor, growth hormone, thyroid stimulating hormone, nerve growthfactor, calcitonin, glucagon and many others, including those presentedin Table 1. In the context of the present invention, receptor mediatedendocytosis affords a convenient mechanism for transporting ananoparticle to the interior of a cell.

In RME, the binding of a ligand by a receptor disposed on the surface ofa cell can initiate an intracellular signal, which can include anendocytosis response. Thus, an agent that is bound on the surface of acell is invaginated and internalized within the cell. Subsequently, anytether sequence present on the nanoparticle can be cleaved by the cell'sendogenous enzymes, thereby freeing the agent to deliver its activeagent to the appropriate structure.

It must be reemphasized that RME is not the exclusive method by which adelivery vehicle can be translocated into a cell. Other methods ofuptake that can be exploited by attaching the appropriate entity to ananoparticle include the advantageous use of membrane pores.Phagocytotic and pinocytotic mechanisms also offer advantageousmechanisms by which a nanoparticle delivery vehicle can be internalized.

VI. Detection of a Nanoparticle Delivery Vehicle

Nanoparticle delivery vehicles of the present invention can be detectedon both the interior and exterior of cells in a variety of ways. Indeed,the ability to select one of several techniques for detection is anaspect of the present invention. One method of detecting the presence ofa nanoparticle delivery vehicle is by monitoring a sample for thehomeostatic change the nanoparticle delivery vehicle is designed toproduce. For some applications, however, it might be desirable tomonitor the presence of a nanoparticle delivery vehicle by a differentapproach. Several, but not all, methods of detecting the presence ofnanoparticle delivery agents can include the use of transmissionelectron, fluorescence and other microscopy techniques;spectroscopic-based detection; and detection methods involving proteins,such as immunological methods. Other methods are possible and willdepend on the specific circumstances of the experiment or treatmentprotocol.

VI.A. Transmission Electron Microscopy Detection of a NanoparticleDelivery Vehicle

Transmission electron microscopy (TEM) can be used to determine thepresence of a nanoparticle delivery vehicle. Nanoparticle deliveryvehicles comprising nanoparticles of 5 nm and larger can be clearlyvisualized by TEM, as evidenced by the TEM images presented in FIGS. 1Aand 1B. FIG. 1A depicts nanoparticle delivery vehicles comprising 5 nmnanoparticles. FIG. 1B depicts nanoparticle delivery vehicles comprising30 nm nanoparticles. In both figures, the nanoparticle delivery vehiclescomprise a nuclear localization signal. FIGS. 1A and 1B indicate thatTEM is a useful method of detecting the presence and subcellularlocalization of nanoparticle delivery vehicles. Nanoparticle deliveryvehicles comprising nanoparticles as small as 5 nm in size are visible,as depicted in FIG. 1A.

FIGS. 1A and 1B demonstrate that TEM can be used to detect the presenceof a nanoparticle delivery vehicle in the nucleus of a cell.Nanoparticle delivery vehicles comprising 5 nm nanoparticles locate tothe cell nucleus, as shown in FIG. 1A. Nanoparticle delivery vehiclescomprising 30 nm nanoparticles remain in the cell's cytoplasm, as shownin FIG. 1B. Thus, TEM facilitates the detection of nanoparticle deliveryvehicles and the subcellular localization of the delivery vehicles.

TEM can also be used to estimate the density of nanoparticle deliveryvehicles in a region. A density calculation can be performed by countingthe number of observed particles in a given area scanned by TEM. Anunderstanding of the density of nanoparticle delivery vehicles in adefined region, such as a cell's nucleus or cytoplasm, can provideinformation regarding the size requirements for a nanoparticle, theeffectiveness of a given nuclear localization signal and otherparameters.

VI.B. Spectroscopic Detection of a Nanoparticle Delivery Vehicle

Nanoparticle delivery vehicles of the present invention can also bedetected spectroscopically. UV, visible and IR spectroscopic methods canbe employed in the present invention. The choice of detection methodwill typically depend on the experimental design. In one embodiment,nanoparticle delivery vehicles of the present invention can beindirectly detected using fluorescence spectroscopy.

Expression of GFP and other fluorescent marker proteins provided by anactive agent of a nanoparticle delivery vehicle of the present inventioncan be detected by fluorescence and can act as an indicator of thepresence of a nanoparticle delivery vehicle. Alternatively, afluorescent moiety can be associated with the nanoparticle component ofa nanoparticle delivery vehicle and in this way, the presence of thenanoparticle delivery vehicle itself can be identified.

VI.C. Microscopy-Based Detection of a Nanoparticle Delivery Vehicle

As noted in section VI.A above, TEM is one form of microscopy useful fordetecting delivery vehicles. Other forms of microscopy, however, canalso be employed. Microscopy techniques such as bright field microscopy,phase contrast microscopy, confocal microscopy and other techniques canbe employed to detect the presence of delivery vehicles.

Phase contrast microscopy is typically used for the visualization ofcellular organelles, and can be employed to detect the presence ofdelivery vehicles. Confocal microscopy can also be useful for detectingdelivery vehicles. The resolution of any of the above microscopytechniques can be enhanced by the introduction of various contrastenhancement or other agents known to refine images and increaseresolution.

VI.D. Protein-Based Detection of a Nanoparticle Delivery Vehicle

Protein-based detection of a nanoparticle delivery vehicle is alsopossible. For example, a second protein known to associate with a firstprotein bound to a nanoparticle can be labeled and used as a probe.Suitable labels include fluorescent moieties and other labels. Uponassociation of the first and second proteins, and therefore associationof the labeled second protein and the nanoparticle delivery vehicle, thepresence of the nanoparticle delivery vehicle is detectable by detectingthe presence of the probe. Any suitable protein pair can be used todetect a nanoparticle delivery vehicle of the present invention;preferably, a first protein is associated with a nanoparticle and asecond protein is labeled with a detectable label, and the two proteinsare known to associate.

VII. Applications of the Nanoparticle Delivery Vehicles of the PresentInvention

The nanoparticle delivery vehicles of the present invention can beemployed to deliver a variety of active agents to a variety of differentcellular and subcellular locations. As described more fully below, thepresent invention is useful for analysis of gene expression;incorporating a nucleic acid sequence or a sequence comprising nucleicacid analogs into a cell's genome; altering the concentration of aregulatory protein in a cell; altering an RNA splicing pattern; andinteracting with mRNA in the cytoplasm of a cell.

As a general rule, when nucleic acid sequences are being selected andmanipulated, care should be taken wherever possible to minimize thepotential for the formation of self-annealed structures. Sequences ofany chemical composition that are predicted to give rise toself-annealing structures should be avoided when practicing the presentinvention.

Additionally, the stringency of hybridization conditions can be varied,with the general rule that the temperature should remain withinapproximately 10° C. of the duplex's predicted T_(m), which is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. An example ofstringent hybridization conditions for analysis of complementary nucleicacids having more than about 100 complementary residues is overnightincubation in 50% formamide with 1 mg of heparin at 42° C. A highstringency wash can be preceded by a low stringency wash to removebackground probe signal. An example of medium stringency wash conditionsfor a duplex of more than about 100 nucleotides is incubation for 15minutes in 1×SSC at 45° C. An example of low stringency wash for aduplex of more than about 100 nucleotides is incubation for 15 minutesin 4-6×SSC at 40° C. For short probes (e.g., about 10 to 50nucleotides), stringent conditions typically involve incubation in saltconcentrations of less than about 1.0 M sodium ion, typically about 0.01to 1.0 M sodium ion (or other ion) concentration, at pH 7.0-8.3, at atemperature of at least about 30° C. Stringent conditions can also beachieved with the addition of destabilizing agents such as formamide. Ingeneral, a signal to noise ratio of 2-fold (or higher) than thatobserved for an unrelated probe in the particular hybridization assayindicates detection of a specific hybridization.

VII.A. Modulation and Analysis of Gene Expression

The nanoparticle delivery vehicles of the present invention can be usedto modulate and analyze gene expression in a model system. It isfundamental that the expression of a gene of interest correlates withthe production of mRNA transcribed from the gene's DNA sequence.Transcribed mRNA is subject to standard Watson-Crick base pairing rules.Thus, in one embodiment a nanoparticle delivery vehicle can be used todirectly modulate expression of a gene by selecting an active agent thatwill remove any expression-inhibiting structures or will introduceexpression-enhancing structures. In another embodiment, a deliveryvehicle of the present invention is employed to mimic a component partof a cell's natural second messenger system and thereby modulate thetranscription and translation of a given gene.

In one embodiment, a transcription factor or other protein having theability to modulate protein expression, which has been presented to thecytoplasm or nucleus of a cell by a nanoparticle delivery vehicle of thepresent invention, modulates gene expression. Modulation encompassesboth up- and downregulation of a gene. In this embodiment, ananoparticle delivery vehicle comprises either a competent transcriptionor translation modulator (e.g., a transcription factor) or a nucleicacid sequence encoding a transcription or translation modulator in therole of active agent. Competent modulators can be active in the form inwhich they are bound to a nanoparticle, or they can be in a form that isactivated by proteolytic or other enzymatic or chemical treatment in thecytoplasm or nucleus of a target cell. Similarly, nucleic acid sequencesencoding a transcription or translation modulator can be translated bythe translation machinery of the target cell, which can be used in amethod analogous to a feedback inhibition loop: the target cell'sexpression machinery can be used to express the nucleic acid introducedby a delivery vehicle, which will then produce a protein can inhibitfurther protein expression.

In another embodiment, gene expression is modulated in a cell havingprotein whose expression is dependent on a given splicing pattern. Inthis embodiment, a nanoparticle delivery vehicle of the presentinvention comprising a morpholino oligonucleotide, PNA or other modifiedoligonucleotide of appropriate sequence that alters splicing, isintroduced to target cells that comprise a gene whose expression isdependent on a splicing event. When a gene encoding green fluorescentprotein (“GFP”) is employed to detectably demonstrate this embodiment ofthe invention, in the absence of such a morpholino oligonucleotide,there is no GFP expression. In cells where the morpholinooligonucleotide delivered by a delivery vehicle of the present inventionis present, alternate splicing events occur and the GFP gene isexpressed and can be detected spectrophotometrically. This example canbe extended to any gene that can be spliced to generate a functionalprotein, with an appropriate nucleotide serving as an active agent boundon a delivery vehicle.

VII.B. Modulating Translation of a Protein

A delivery vehicle of the present invention can be used to deliver anucleic acid sequence for incorporation into the genome of a targetcell. This concept is sometimes referred to as “antisense” or “genetherapy”. Breakthroughs in molecular biology and the Human GenomeProject have opened previously unforeseen possibilities for targetedintervention with gene expression. These include permanent approachessuch as transgenic overexpression or recombinant disruption of specificgenes, as well as novel approaches for transient suppression of genefunction. Short synthetic antisense (AS) oligodeoxynucleotides (ODN)designed to hybridize with specific sequences within a targeted mRNAbelong to the latter class. Integration of a lacking gene into a host'sgenome has also been of significant interest.

AS intervention in the expression of specific genes can be achieved bythe use of synthetic antisense oligodeoxynucleotides (AS-ODNs). Seegenerally, Agrawal, (1996) Trends Biotechnol. 14(10): 376-87; Lev-Lehmanet al., (1997) in Antisense Therapeutics, (Cohen & Smisek, eds.), PlenumPress, New York; and Lefebvre-D'Hellencourt et al., (1995) Eur. CytokineNetw., 6: 7-19; Oligonucleotide & Gene Therapy—Base AntisenseTherapeutics, (1997), (Mori, ed.), Drug & Market DevelopmentPublications, Westborough, Mass.; Antisense Therapeutics, (1996)(Agrawal, ed.), Humana Press, Totowa, N.J. for general antisensereviews. AS-ODNs are short sequences of DNA, typically 15 to 25 bases inlength, and are designed to complement a target mRNA of interest and toform an RNA:ODN duplex. This duplex formation can prevent processing,splicing, transport or translation of the relevant mRNA. Moreover,certain AS-ODNs can elicit cellular RNase H activity when hybridizedwith their target mRNA, resulting in mRNA degradation. Calabretta etal., (1996) Semin. Oncol., 23: 78-87. In that case, RNase H will cleavethe RNA component of the duplex and can potentially release the AS-ODNto further hybridize with additional molecules of the target RNA. Anadditional mode of action results from the interaction of AS-ODNs withgenomic DNA to form a triple helix that might be transcriptionallyinactive.

The nanoparticle delivery vehicles of the present invention can be usedto vary a target cell's expression profile using the above discussion asa general guide. In one embodiment, a nanoparticle delivery vehicle canbe prepared that comprises at least one AS sequence, an ODN sequence, anAS-ODN sequence or other nucleic acid or modified nucleic acid sequence.This sequence can serve as an active agent in a nanoparticle deliveryvehicle. The nanoparticle delivery vehicle also comprises a nuclearlocalization signal, which targets the delivery vehicle to the nucleusof a cell. Alternatively, if it is desired that the nanoparticledelivery vehicle remain in the cytoplasm, nanoparticles of larger size(e.g., 30 nm) can be employed. The precise sequence and/or compositionof an active agent reflects the role or roles of the nanoparticledelivery vehicle. For example, an active agent can be complementary to agene of interest.

In practice, a nanoparticle delivery vehicle designed to vary a targetcell's protein translation profile can be administered to a subject byinjection or, if the target cells are present in an in vitroenvironment, the nanoparticle delivery vehicle can be added directly tothe cell's growth medium. Both introduction procedures are describedherein above, and others will be apparent to one of skill in the artupon review of the present disclosure.

VII.C. Modulating Regulatory Protein Concentration

The nanoparticle delivery vehicles of the present invention can be usedto modulated the concentration of various cellular regulatory proteins.A specific example of regulation is known as using a transcriptionfactor decoy. As stated herein above, the terms “decoy” and“transcription factor decoy” refer to molecules that bind to or interactwith transcription factors and prevent their binding to native enhancersequences. It is possible to design transcription factor decoys thatspecifically interact with transcription factors and mimic or resemblethe natural genomic binding site for the particular transcriptionfactor. Some transcription factor decoys can bind the transcriptionfactor with an affinity near or exceeding its affinity for the naturalgenomic binding site.

Some transcription factors, in addition to binding an endogenous genomicbinding site, can also bind to intracellular soluble ligands. Binding ofsuch a transcription factor to an appropriate ligand subsequently altersthe binding profile of the transcription factor to its genomic bindingsite or sites. Restated, ligand binding by a transcription factor canmodulate the ability of the transcription factor to bind its intendedgenomic site. Such transcription factors are referred to in the art asintracellular or nuclear receptors for soluble ligands.

Transcription factor decoys can function in a variety of ways and thuscan comprise a variety of elements. For example, nucleic acid sequencescan compete with cellular target DNA for binding to one or moretranscription factors. In this example, nucleic acid sequences can forma duplex with a target sequence and effectively inactivate the sequence.Nucleic acid sequences can be introduced that will form otherduplex-type structures such as hairpins, cruciforms or other structuresthat will effectively inactivate cellular target DNA.

There is no requirement that a sequence introduced to cellular targetDNA necessarily comprise unmodified nucleic acids. Sequences cancomprise nucleic acid molecules that contain modified phosphodiesterbonds. Modified phosphodiester bonds can include phosphorothioate,phosphoramidite, and methyl phosphate derivatives, for example.

The nanoparticle delivery vehicles of the present invention are able tofacilitate a modulation in regulatory protein concentration. Modulationcan be achieved by associating an appropriate active agent with ananoparticle. For example, a short sequence of double stranded DNA, forwhich a given regulatory protein (e.g., transcription factor) has a highaffinity, can be used as a transcription factor decoy, as describedherein above. Additional regulatory protein-modulating applications fora delivery vehicle of the present invention will be apparent to one ofskill in the art when considered in view of the present disclosure.

VII.D. Modulating RNA Splicing

Generally, the expression of a specific gene can be regulated at anystep in the process of producing an active protein. Modulation of totalprotein activity can occur via transcriptional, transcript-processing,translational or post-translational mechanisms. One role of ananoparticle delivery vehicle of the present invention is to modulatetranscription of a nucleic acid sequence.

Transcription means a cellular process involving the interaction of anRNA polymerase with a gene that directs the expression as RNA of thestructural information present in the coding sequences of the gene. Theprocess includes, but is not limited to the following steps: (1)transcription initiation, (2) transcript elongation, (3) transcriptsplicing, (4) transcript capping, (5) transcript termination, (6)transcript polyadenylation, (7) nuclear export of the transcript, (8)transcript editing, and (9) stabilizing the transcript. Transcriptioncan be modulated by altering the rate of transcriptional initiation orthe progression of RNA polymerase (Maniatis et al., (1987) Science, 236:1237-45). Transcript-processing can be influenced by circumstances suchas the pattern of RNA splicing (the splicing of the RNA to yield one ormore mRNA species), the rate of mRNA transport to the cytoplasm or mRNAstability.

Additionally, although some eukaryotic mRNA transcripts are directlytranslated, many contain one or more regions, known as “introns,” whichare excised from a transcript before it is translated. The remaining(and therefore translated) regions are known as “exons” and are splicedtogether to form a continuous mRNA sequence. mRNA splice sites, i.e.,intron-exon junctions, can be target regions for an active agent of thepresent invention, and can be particularly useful in situations whereaberrant splicing is implicated in disease, or where an overproductionof a particular mRNA splice product is implicated in disease. Aberrantfusion junctions due to rearrangements or deletions can also be targets.

The present invention can be employed to modulate RNA splicing. Thisaspect of the present invention can be accomplished by selecting anappropriate active agent, such as a nucleic acid sequence known to alterthe splicing pattern for a given gene. The use of an appropriatesequence (e.g., DNA, RNA, morpholino nucleotide, etc.) can influence thesplicing pattern and consequently the protein expression profile for acell. Delivery of the appropriate sequence is the major obstacle intherapeutic applications of RNA splicing that can be overcome byemploying nanoparticle delivery vehicles comprising a nuclear targetingcapability.

VII.E. Interaction with mRNA in the Cytoplasm

The present invention can be used to interact with mRNA transcript for agiven protein while the transcript is in the cytoplasm. Interaction cantake a variety of forms, including modulation of the amount of a givenprotein produced by a cell. In one aspect of the present invention, ananoparticle delivery vehicle of the present invention can employ anantisense nucleotide to interact with mRNA which has been exported tothe cytoplasm. See, e.g., Bassell et al., (1999) FASEB J. 13: 447-54.

A nanoparticle delivery vehicle of the present invention can be designedto interact with mRNA in the cytoplasm of a cell. The specifichybridization of an oligomeric compound with mRNA can interfere with thenormal function of the mRNA. This modulation of function of a nucleicacid by compounds that specifically hybridize to it is generallyreferred to as “antisense” and is discussed herein above. Antisensecompounds can interrupt various functions of RNA can include all vitalfunctions such as, for example, translocation of the RNA to the site ofprotein translation, translation of protein from the RNA, splicing ofthe RNA to yield one or more mRNA species, and catalytic activity whichmight be engaged in or facilitated by the RNA. The overall effect ofsuch interference with mRNA function is the modulation of the expressionof the protein for which the mRNA codes.

Association of an antisense compound with, for example, mRNA, can beutilized for diagnostics, therapeutics, prophylaxis and as researchreagents and kits. When used as a therapeutic, a subject suspected ofhaving a disease or disorder that can be treated by modulating theexpression of a given protein can be treated by administering ananoparticle delivery vehicle of the present invention comprising anantisense active agent. Use of the nanoparticle delivery vehicles of thepresent invention, when antisense compounds are included as the activeagent, can also be used prophylactically, e.g., to prevent or delayinfection, inflammation or tumor formation, for example.

Antisense-bearing delivery vehicles of the present invention are usefulfor research and diagnostics, because the antisense component canhybridize with nucleic acids encoding a particular protein of interest,enabling sandwich and other assays to easily be constructed to exploitthis fact.

Additionally, the nanoparticle delivery vehicles of the presentinvention can be localized subcellularly through the selection of anappropriately sized nanoparticle. As shown in FIG. 1B, delivery vehiclescomprising nanoparticles of 30 nm or greater remain localized to thecytoplasm of a cell and are not localized in the cell's nucleus,regardless of the presence or absence of a nuclear localization signal.This observation indicates that larger nanoparticles can be targeted tothe cytoplasm, wherein untranslated mRNA is localized. Smallernanoparticles can be used to target pre-mRNA in the nucleus. Thus, adelivery vehicle of the present invention can be targeted to thecytoplasm of a cell, where it can interact with RNA sequences disposedtherein.

VIII. Advantages of the Delivery Vehicles of the Present Invention

There are a number of advantages of the present invention over themethods for delivery presently known in the art. First, there is adistinct advantage in the use of a nuclear localization signal. Theinclusion of an NLS as a component part of a nanoparticle deliveryvehicle assures that, assuming optimization of other variables, thenanoparticle delivery vehicle is targeted directly to the nucleus of acell. This advantage greatly increases the efficacy of an active agentdesigned to interact with nuclear structures by increasing the amount ofmaterial delivered to the nucleus of a target cell; inclusion of the NLSresults in less material disposed in the cytoplasm for shorter periodsof time and ultimately less degradation of material. The NLSadditionally offers the ability to effectively target cellular processesthat occur in the nucleus, such as DNA replication, transcription, andvarious splicing events.

The association of additional targeting agents can aid in thetranslocation of a vehicle across various membranes, such as the nuclearmembrane of a cell or the outer membrane of a cell, thus providinganother advantage. If membranes and other structures that generallyinhibit translocation of a vehicle to a given location in or on a cellare analogized as “locks”, NLS and RME sequences can be analogized to be“keys”. Thus, in a preferred embodiment, a nanoparticle delivery vehicleof the present invention can comprise a plurality of different sequencesor “keys,” which can enable a given nanoparticle delivery vehicle topass through various potential barriers to translocation.

Another advantage of a preferred embodiment of the nanoparticle deliveryvehicles of the present invention is the ability to create nanoparticlesthat comprise a material that is biologically inert. For example, it ispossible to fashion nanoparticles from gold and other materials. Gold,unlike some other materials, is biologically inert and can bephysiologically tolerated without significant adverse effects bybiological systems. Further, a nanoparticle can comprise biodegradablematerial, which upon breakdown, can yield the nanoparticle deliveryvehicle's component parts, all of which are themselves biodegradable.

Yet another advantage of a preferred embodiment of the nanoparticledelivery vehicles of the present invention is their ability to functionin any of a variety of roles, due to the lack of restriction on theactive agent. A nanoparticle delivery vehicle of the present inventioncan therefore fill a variety of roles by simply changing the activeagent to suit the need. Thus, a nanoparticle delivery vehicle designedto modulate gene expression by delivering an antisense strand to thenucleus of a cell can also function as a transcription factor decoy byreplacing the antisense strand active agent with a double strandedsequence of DNA.

Finally, the size of the nanoparticle can be varied, which can providefor differential targeting of a nanoparticle delivery vehicle.Nanoparticle size can influence the targeting of a delivery vehicle. Ananoparticle delivery vehicle comprising a nanoparticle of about 30 nmor larger will not be transported into the nucleus of a cell and willremain in the cytoplasm of the cell, even if an NLS is present. However,although such a nanoparticle delivery vehicle might not be transportedto the nucleus of a cell, the nanoparticle delivery vehicle can beinternalized by the cell and remain localized in the cytoplasm. Thus,such vehicles can be useful for modulating processes occurring in thecytoplasm, such as translation and translocation.

Laboratory Example

The following Laboratory Example has been included to illustratepreferred modes of the invention. Certain aspects of the followingLaboratory Example are described in terms of techniques and proceduresfound or contemplated by the present inventors to work well in thepractice of the invention. This Laboratory Example are exemplifiedthrough the use of standard laboratory practices of the inventors. Inlight of the present disclosure and the general level of skill in theart, those of skill will appreciate that the following LaboratoryExample is intended to be exemplary only and that numerous changes,modifications and alterations can be employed without departing from thespirit and scope of the present invention.

Laboratory Example

Targeted entry into cells is an increasingly important area of research.The nucleus is a desirable target since the genetic information of thecell and transcription machinery resides there. The diagnoses of diseasephenotype, the identification of potential drug candidates, and thetreatment of disease by novel methods such as antisense therapy would beenhanced greatly by the efficient transport of materials to living cellnuclei (Kole & Sazani, (2001) Curr. Opin. Mol. Ther. 3: 229-234). Theintracellular fate of gold nanoparticles chemically designed to transitfrom outside a living cell into the nucleus is reported in the presentLaboratory Example.

Although metal, semiconductor, polymer, and magnetic particles have beenintroduced into cells previously (Liu et al., (2001) Biomacromolecules2: 362-368; Marinakos et al., (2001) J. Phys. Chem. B. 105: 8872-8876;West & Halas, (2000) Curr. Opin. Biotech. 11: 215-217; Hogemann et al.,(2002) Bioconjugate Chem. 13: 116-121), there is no comprehensivecytochemical approach to targeting the nucleus from outside the plasmamembrane of living cells. The development of an approach that permitsthe transport of nanometer-sized particles into cells has importantapplications in cell biology as a tool for the study of cell developmentand differentiation.

A number of techniques have been used previously to determine cellulartrajectories of particles. Indeed, the use of electron microscopy withcolloidal gold stains was perhaps the first modern method of cellstructure characterization (Havat (Ed.), (1998) Colloidal Gold.Principles, Methods, and Applications; Academic Press, Inc.: San Diego,Vol. 1). More recently, fluorescence microscopy has been used to locatefluorophores, including luminescent CdSe nanoparticles in cells (Bruchezet al., (1998) Science 281: 2013-2016; Nie & Chan, (1998) Science 281:2016-2018). However, prior studies of nuclear translocation ofnanoparticles were performed using microinjection or chemically modifiedcells, thus bypassing cellular membrane entry. The combination oftargeted endocytosis coupled with nuclear uptake has not beendemonstrated in a nanoparticle vector using intact cells, prior to thepresent disclosure.

Targeted nuclear delivery is a challenging task, as any cell-specificnuclear probe must satisfy minimally the following requirements(Hallenbeck & Stevenson, (2000) Targetable Gene Delivery Vectors (Habib,Ed.), Kluwer Academic/Plenum Publishers: New York, pp 37-46): it must(i) be small enough to enter the cell and cross the nuclear membrane;(ii) bind to cell-specific plasma membrane receptors byreceptor-mediated endocytosis (RME), for example; (iii) escapeendosomal/lysosomal pathways; (iv) pass through the nuclear porecomplex, and (v) have low toxicity. In the present Laboratory Example,results of intracellular trafficking studies of nanoparticles designedto perform these and other functions are reported.

A nanoparticle vector of the present Laboratory Example comprises a coreof a 20 nm gold particle and a shell of bovine serum albumin (BSA)conjugated to various cellular targeting peptides, which are presentedin the following Table of Representative Peptide Sequences:

Table of Representative Peptide Sequences Peptide Sequence SEQ ID NOSource Peptide/BSA N0 CGGGPKKKRKVGG 3 SV40 large T NLS 7 ± 1 N1CGGFSTSLRARKA 4 Adenoviral NLS 8 ± 1 N2 CKKKKKKSEDEYPYVPN 5 AdenoviralRME 9 ± 2 N3 CKKKKKKKSEDEYPYVP 6 Adenoviral Fiber Protein 6 ± 2NFSTSLRARKAWhen preparing the peptides disclosed in the Table of RepresentativePeptide Sequences, peptides were conjugated to BSA with a 3-maleimidobenzoic acid N-hydroxysuccinimide ester linker. Gel electrophoresis(SDS-PAGE and IEF) was used to quantify peptide:BSA ratio. Each peptidewas chosen to perform a certain task (e.g., RME). Individual peptideshave been explored previously as therapeutic delivery vectors (Morris etal., (2000) Curr. Opin. Biotech. 11: 461-466). However, highly efficientnuclear targeting in biology is accomplished by viruses, which utilizedifferent peptides for each barrier mentioned above. A significantobservation of the present Laboratory Example is that viral peptidesconjugated to proteins on the surface of a nanoparticle retain theirfunction of promoting cell entry and nuclear targeting. Moreover,separate short peptides on a single particle lead to more efficientnuclear targeting than a single long peptide. Together the gold core andmultifunctional peptide shell provides a flexible scaffold that can betuned to target specific cells for intranuclear assays or therapeuticdelivery.

Gold was chosen as an intracellular targeting vector primarily for threereasons. First, gold can be routinely synthesized in sizes varyingcontinuously from 0.8 nm to 200 nm with <5% size dispersity. Secondly,gold can be modified with a large collection of small molecules,peptides, proteins, DNA, and polymers. Moreover, all of these functionalelements can be combined on a single particle, often via simple one-potprocedures. Finally, gold particles have strong visible lightextinctions that can be used to monitor their trajectories inside cellsunder polarized light conditions. These properties were advantageouslyemployed in a novel combination of video-enhanced color (VEC) microscopyand differential interference contrast microscopy (DIC), whichfacilitated the observation of the trajectory of 20 nm goldnanoparticles inside cells.

Dynamic light scattering and transmission electron microscopy revealedthat BSA-peptide conjugates add <2 nm to the radius of the nanoparticlecomplex. The fact that BSA does not add greatly to the size of the goldparticle is important in its use in constructing nuclear targetingvectors because the diameter of the nuclear pore complex is 20-50 nmdepending on the cell line (Feldherr & Akin, (1990) J. Cell Biol.111:1-8). The 20 nm gold particles used in he present Laboratory Examplehave a maximum diameter of 25 nm when complexed with any of theBSA-peptide conjugates studied (see Supporting Information).

Nuclear translocation through the nuclear pore complex has previouslybeen studied using gold nanoparticles labeled with an NLS from SV-40virus (large T antigen). In the classic studies nuclear targeting wasobserved by transmission electron microscopy (TEM) followingmicroinjection into the cell (Feldherr et al., (1992) Proc. Nat. Acad.Sci. U.S.A. 89: 11002-11005). As a test case, nanoparticle complexescomprising peptide N0 were introduced into the growth medium of HepG2cells. Surprisingly, N0 complexes were observed inside the cytoplasm ofHepG2 cells, however N0 did not enter the nucleus. Experiments at 4° C.indicated that cell entry was via an energy-dependent pathway. Thisobservation suggests that N0 entered the cell by receptor-mediatedendocytosis, but was unable to escape the endosome and target thenucleus (TEM and confocal fluorescence microscopy confirmed thatnanoparticles were confined to endosomes). These results highlight thechallenges associated with nuclear targeting: although a known NLSpeptide is able to enter HepG2 cells, it cannot target the nucleusunless it is capable of endosomal escape.

In an effort to enhance nuclear targeting efficiency in HepG2 cells,peptides from the adenovirus were explored. The adenovirus is widelyused in gene delivery and there is a great deal of interest in replacingthe whole virus, which is potentially infectious and immunogenic, withpeptide sequences derived from the adenovirus fiber protein (Seth,(2000) Adenoviral Vectors; (Habib, Ed.) Kluwer Academic/PlenumPublishers: New York, pp 13-22; Bilbao et al., (1998) TargetedAdenoviral vectors for Cancer Gene Therapy, Plenum Press: New York, Vol.57, pp 365-374). This protein is known to contain both RME and NLSsequences (N1 and N2, in the Table of Representative Peptide Sequences).The full length fiber containing both the RME and NLS is peptide N3 inthe Table of Representative Peptide Sequences. A comparison of thefunctions of these targeting peptides when complexed to a goldnanoparticle is as follows. N1 does not enter the cell. N2 enters thecell, but remains trapped in endosomes and does not reach the nucleus.N3 targets the nucleus, however, N1/N2 has a greater propensity fornuclear targeting than N1, N2, or N3. These results are interpreted asfollows. N1 presents only the adenovirus NLS (Table of RepresentativePeptide Sequences). This peptide does not function as an RME and has noother chemical moiety that permits cell entry. N2 presents the RME (NPXY(SEQ ID NO: 7) motif) and it enters the cell, however, it is not capableof nuclear targeting (Chen et al., (1990) J. Biol. Chem. 265:3116-3123).

These results show that the nanoparticle complex must present both RMEand NLS in order to both enter the cell and achieve nuclearlocalization. The VEC-DIC results clearly show significant numbers of N3in the nucleus in agreement with a gene delivery study using thispeptide (Zhang et al., (1999) Gene Ther. 6: 171-181). The N1/N2-labelednanoparticle is even more efficient, as seen by VEC-DIC microscopy.

Another comparison to be made is between a multi-functional nanoparticleN1/N2 that presents the RME and NLS on separate BSA bioconjugates andN3, which presents the full-length adenoviral fiber peptide. N1/N2 waspresent in the nucleus in greater numbers than N3. The origin of thehigher nuclear targeting efficiency in particles carrying two shortpeptides versus one long sequence could be structural or spatial.Infrared spectroscopy indicates that all peptides employed in thisExa,[;e adopt an extended confirmation when attached to nanoparticles.However, when one long peptide is synthesized with two consecutivesignals, it is likely that one of the signals will be less accessible tocellular receptors. This is important for NPXY (SEQ ID NO: 7) motifs,for example, since tandem interaction of two NPXY (SEQ ID NO: 7) regionshas been shown to facilitate RME (Hussain, (2001) Front. Biosci. 6:417-428). Attaching the two-peptide signals to a nanoparticle asseparate, shorter pieces likely gives them equal access to cellularreceptors.

The methods used here provide an approach for rapidly assessing theefficacy of various combinations of targeting peptides usingnanoparticle complexes for nuclear targeting. The VEC-DIC combinationmicroscopy permits examination of hundreds of samples per day, animprovement over costly and time consuming electron and confocalmicroscopy techniques.

The multifunctional approach demonstrated using adenoviral targetingsequences provides a test of the function of individual peptidesequences that will permit effective and cell-specific targeting for arange of scientific and medical applications.

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The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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It will be understood that various details of the invention can bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only, andnot for the purpose of limitation—the invention being defined by theclaims.

1. A nanoparticle delivery vehicle for contacting a target cell andcrossing a cellular membrane and a nuclear membrane of a target cell,the nanoparticle delivery vehicle comprising: (a) a nanoparticle,wherein the nanoparticle comprises a material selected from the groupconsisting of cadmium selenide, titanium, titanium dioxide, tin, tinoxide, silicon, silicon dioxide iron, iron^(III) oxide, silver, nickel,gold, copper, aluminum, steel, cobalt-chrome alloy, titanium alloy,brushite, tricalcium phosphate, alumina, silica, zirconia, diamond,polystyrene, silicone rubber, polycarbonate, polyurethanes,polypropylenes, polymethylmethaacrylate, polyvinyl chloride, polyesters,polyethers, and polyethylene; (b) an active agent; (c) a nuclearlocalization signal; and (d) one or more extracellular targeting agents,wherein the one or more extracellular targeting agents is recognized andbound by one or more receptors on a surface of a target cell.
 2. Thenanoparticle delivery vehicle of claim 1, wherein the nanoparticle isbiodegradable.
 3. The nanoparticle delivery vehicle of claim 1, whereinthe nanoparticle ranges from about 1 nm to about 1,000 nm in diameter.4. The nanoparticle delivery vehicle of claim 1, wherein thenanoparticle is about 30 nm or less in diameter.
 5. The nanoparticledelivery vehicle of claim 1, wherein the active agent is selected fromthe group consisting of oligomers of double stranded nucleic acids,single stranded nucleic acids, chemically modified nucleic acids,peptide nucleic acids, proteins and small molecules.
 6. The nanoparticledelivery vehicle of claim 1, further comprising a tether sequenceattached to, and disposed between, the active agent and thenanoparticle.
 7. The nanoparticle delivery vehicle of claim 1, whereinthe nanoparticle delivery vehicle is disposed in a pharmaceuticallyacceptable diluent.
 8. The nanoparticle delivery vehicle of claim 1,further comprising a detectable moiety.
 9. The nanoparticle deliveryvehicle of claim 8, wherein the detectable moiety is a fluorescentcompound.
 10. The nanoparticle delivery vehicle of claim 1, wherein eachsequence is independently associated with the nanoparticle.
 11. Thenanoparticle delivery vehicle of claim 1, wherein the extracellulartargeting agent is an RME motif.
 12. The nanoparticle delivery vehicleof claim 1, wherein the extracellular targeting agent is selected fromthe group consisting of diptheria toxin, pseudomonas toxin, choleratoxin, ricin, concanavalin A, Rous sarcoma virus, Semliki forest virus,vesicular stomatitis virus, adenovirus, transferrin, low densitylipoprotein, transcobalamin, yolk proteins, IgE, polymeric IgA, maternalIgG, insulin, epidermal growth factor, growth hormone, thyroidstimulating hormone, nerve growth factor calcitonin, glucagon,prolactin, luteinizing hormone, thyroid hormone, platelet derived growthfactor, interferon, nuclear localization signal and catecholamines. 13.The nanoparticle delivery vehicle of claim 1, wherein the extracellulartargeting agent comprises a fragment of a molecule selected from thegroup consisting of diptheria toxin, pseudomonas toxin, cholera toxin,ricin, concanavalin A, Rous sarcoma virus, Semliki forest virus,vesicular stomatitis virus, adenovirus, transferrin, low densitylipoprotein, transcobalamin, yolk proteins, IgE, polymeric IgA, maternalIgG, insulin, epidermal growth factor, growth hormone thyroidstimulating hormone, nerve growth factor, calcitonin, glucagon,prolactin, luteinizing hormone, thyroid hormone, platelet derived growthfactor, interferon and catecholamines.
 14. The nanoparticle deliveryvehicle of claim 1, further comprising two or more different activeagents.
 15. The nanoparticle delivery vehicle of claim 1, furthercomprising a biocompatibility-enhancing agent.
 16. The nanoparticledelivery vehicle of claim 1, further comprising a protective coatingcovering at least part of the delivery vehicle.
 17. The nanoparticledelivery vehicle of claim 16, further comprising a protective coatingcovering the entire delivery vehicle.
 18. The nanoparticle deliveryvehicle of claim 16, wherein the protective coating comprises a polymer.19. The nanoparticle delivery vehicle of claim 16, wherein theprotective coating comprises a biological material.
 20. The nanoparticledelivery vehicle of claim 16, wherein the biological material is aprotein, lipid, carbohydrate, or combination thereof.
 21. A nanoparticledelivery vehicle for contacting a target cell and crossing a cellularmembrane and a nuclear membrane of a target cell, the nanoparticledelivery vehicle comprising: (a) a nanoparticle, wherein thenanoparticle comprises a material selected from the group consisting ofcadmium selenide, titanium, titanium dioxide, tin, tin oxide, silicon,silicon dioxide, iron, iron^(III) oxide, silver, nickel, gold, copper,aluminum, steel, cobalt-chrome alloy, titanium alloy. brushite,tricalcium phosphate, alumina, silica, zirconia, diamond, polystyrene,silicone rubber, polycarbonate, polyurethanes, polypropylenes,polymethylmethaacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene; (b) an active agent; (c) a nuclear localization signal;and (d) one or more extracellular targeting agents, wherein the one ormore extracellular targeting agents is recognized and bound by one ormore receptors on a surface of a target cell; wherein the nuclearlocalization signal or one of the one or more extracellular targetingagents is selected from the group consisting of SEQ ID NOs: 4-6, andcombinations thereof.
 22. A nanoparticle delivery vehicle for contactinga target cell and crossing a cellular membrane and a nuclear membrane ofa target cell, the nanoparticle delivery vehicle comprising: (a) ananoparticle, wherein the nanoparticle comprises a material selectedfrom the group consisting of cadmium selenide, titanium, titaniumdioxide, tin, tin oxide, silicon, silicon dioxide, iron, iron^(III)oxide, silver, nickel, gold, copper, aluminum, steel, cobalt-chromealloy, titanium alloy, brushite, tricalcium phosphate, alumina, silica,zirconia, diamond, polystyrene, silicone rubber, polycarbonate,polyurethanes, polypropylenes, polymethylmethaacrylate, polyvinylchloride, polyesters, polyethers, and polyethylene; (b) an active agent;(c) a nuclear localization signal; and (d) one or more extracellulartargeting agents, wherein the one or more extracellular targeting agentsis recognized and bound by one or more receptors on a surface of atarget cell; wherein the nuclear localization signal comprises SEQ IDNO: 4 and one of the one or more extracellular targeting agentscomprises SEQ ID NO:
 5. 23. The nanoparticle delivery vehicle of claim22, wherein the target cell is a human hepatocarcinoma cell.
 24. Ananoparticle delivery vehicle for contacting a target cell and crossinga cellular membrane and a nuclear membrane of a target cell, thenanoparticle delivery vehicle comprising: (a) a nanoparticle, whereinthe nanoparticle comprises a material selected from the group consistingof cadmium selenide, titanium, titanium dioxide, tin, tin oxide,silicon, silicon dioxide, iron, iron^(III) oxide, silver, nickel, gold,copper, aluminum, steel, cobalt-chrome alloy, titanium alloy, brushite,tricalcium phosphate, alumina, silica, zirconia, diamond, polystyrene,silicone rubber, polycarbonate, polyurethanes. polypropylenes,polymethylmethaacrylate, polyvinyl chloride, polyesters, polyethers, andpolyethylene; (b) an active agent; (c) a nuclear localization signal;and (d) one or more extracellular targeting agents, wherein the one ormore extracellular targeting agents is recognized and bound by one ormore receptors on a surface of a target cell; wherein the nuclearlocalization signal and one of the one or more extracellular targetingagents together comprise SEQ ID NO:
 6. 25. The nanoparticle deliveryvehicle of claim 24, wherein the target cell is a human hepatocarcinomacell.
 26. A nanoparticle delivery vehicle for contacting a target celland crossing a cellular membrane and a nuclear membrane of a targetcell, the nanoparticle delivery vehicle comprising: (a) a nanoparticle;(b) an active agent; (c) a nuclear localization signal; and (d) one ormore extracellular targeting agents, wherein the one or moreextracellular targeting agents is recognized and bound by one or morereceptors on a surface of a target cell; wherein the nuclearlocalization signal or one of the one or more extracellular targetingagents is selected from the group consisting of SEQ ID NOs: 4-6, andcombinations thereof.